Histology and Cytology

NTA Level 5

Session 02: Tissue fixation

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 Learning objectives
• By the end of this session, students should be able to:
• Define fixation, putrefaction and autolysis
• Describe postmortem changes
• Explain putrefaction and autolysis
• Describe signs of autolysis
• State the criteria of a good fixative
• Outline the purpose of fixation
• Classify fixatives into simple and compound fixatives
• Define micro-anatomical and cytological fixatives
• Differentiate nuclear fixatives and cytoplasmic fixatives 2
 Learning objectives…

• Explain the treatment procedures of tissues after

fixing in various fixatives
• Explain the preservation and storage procedures
of tissues
• Describe common fixation artifacts
• Define common terms applied in fixation of
tissues

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 Activity: Brainstorming

• How do you understand about tissue fixation?

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 Definition of Terms

Fixation
• Is the preservation of biological tissues from decay due
to autolysis and putrefaction.
• The chemical substance used to achieve this is called
fixative.
Fixative
• Is a chemical substance that will preserve after death
the shape, structure, relationship and chemical
constituents of tissues and cells in as life- manner as
possible. 5
 Definition of Terms…

Post mortem changes

• These are changes that take place on tissue
cells after the death of the body or after the
tissue is removed from the body.
There are two changes namely;
• Putrefaction
• Autolysis

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 Definition of Terms…

Putrefaction
• Is the breakdown of tissue cells after death due
to bacterial action often with formation of gas.
The bacteria disseminate from the alimentary
tract and the surrounding organs.
• During life these bacteria are commensals.

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 Definition of Terms…

Autolysis
• Is the dissolving or lysing of cells due to enzymatic action.
After death the normal behavior of enzymes is altered.
• The lysosome ruptures to release enzymes which
dissolve the surrounding cells. This change is prominent
in specialized organs like the brain, liver, kidney and the
alimentary canal.
• This is caused by a group of enzymes mainly
cathepsins, some of which shorten proteins to peptides
and some are carboxy peptidases and aminopeptidases
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which break down peptides to individual amino acids.
 Definition of Terms

Note
• High temperatures increases the rate of
autolysis but reduces fixation time.
• The optimum temperature for autolysis and
putrefaction is 37oC.
• Autolysis and putrefaction are arrested by deep
freezing and reduced to minimum by
refrigeration at 4oC.
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 Signs of autolysis

• During autolysis various parts of the cell undergo

different changes. For example;
The nucleus
• It first condenses (pyknosis), then fragments
(karyorrhexis) and eventually disappears
(karyolysis).
The cytoplasm
• It swells, becomes granular and eventually
becomes a homogenous mass with the loss of the 10

normal staining reaction.

 Signs of autolysis…

The epithelium
• It is desquamates and splits away from the
basement membranes
The glycogen
• It diminishes or diffuses out of the, leaving
empty spaces.

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 Fixation

• Fixation should be carried out as soon as

possible after removal of the tissues (in the case
of surgical pathology) or soon after death (with
autopsy) to prevent autolysis.
• The choice of fixative depends on the staining
technique (histological staining,
immunohistochemistry, in situ hybridization) to be
used later.
• Most histological staining methods, but not all, 12

allow the use of formalin.

 Fixation…

• Immunohistochemistry can be performed on

paraffin/plastic embedded or cryopreserved
specimens that have been fixed, but some antibodies
will not be able to bind to the target molecules after
the formalin fixation and paraffin embedding
processes.
• This process provides rigidity to the tissue, making it
easier to section.
• There are a variety of forms of fixation, including
"fresh frozen," where the tissue is not actually placed 13

in fixative
 Fixation…

• There is no perfect fixative, though

formaldehyde comes the closest.
• Therefore, a variety of fixatives are available for
use, depending on the type of tissue present
and features to be demonstrated

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 Type of Fixation

• Immersion fixation
• Perfusion fixation
• Vapour fixation
• Coating/Spray fixation
• Freeze drying
• Microwave fixation/Stabilization

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 Immersion fixation
• This is the commonest way of fixation in the
laboratories. In this technique the whole
specimen is immersed in the liquid fixative such
as tissue samples are immersed in 10% neutral
buffered formalin or cytology smear in 95% ethyl
alcohol.

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 Perfusion fixation
• This is mainly used in research purpose. In this
technique the fixative solution is infused in the
arterial system of the animal, and the whole
animal is fixed.
• The organ such as the brain or spinal cord can
also be fixed by perfusion fixation.

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 Vapour fixation
• In this type of fixation, the vapour of chemical is
used to fix either a smear or tissue section.
• The commonly used chemicals for vapour fixation
are formaldehyde, osmium tetroxide,
glutaraldehyde and ethyl alcohol.
• The vapour converts the soluble material to
insoluble material, and these materials are
retained when the smear comes in contact with
liquid solution. 18
 Coating/Spray fixation
• This is commonly used in the cytology samples.
The spray fixative is used for easy transportation
of the slide.
The main advantages of spray fixatives are:
(a) Fixation of the cells
(b) To impart a protective covering over the smear
(c) No need to carry liquid fixative in bottle or jar

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 Criteria of a good fixative

• It must cause sudden death of tissue cells

• It must be antibacterial
• It must inactivate enzymes
• It must penetrate the tissue quickly and evenly
• It must render insoluble substances of the cells
• It must preserve cells in as life-like manner as
possible
• It must harden tissue and enable easy manipulation
of naturally soft tissues of organs such as brain, 20

liver and spleen.

 Criteria of a good fixative…

• It must solidify colloidal material to irreversible

semi- solid state that is easily demonstrated.
• It must alter to varying degrees the refractive
index indices of different cell structures so that
they are made more visible due to differentiation.
• It must fortify the tissue against the harsh effects
of solutions used for processing of tissues.
• It must have a good effect on staining
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 Criteria of a good fixative…

Note
• The amount of fixatives should be 15-20 times
the bulk of the tissue to be fixed.
• Thin slices of tissue should be made to enable
quick penetration and even fixation of tissue. In
cases where broad or whole organs are to be
fixed for processing, it must be ensured that
they are trimmed then. i.e. 3-5mm thick
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 Purpose of Fixation
1. To preserve tissue components permanently in as
life-like a state as possible.
2. To prevent autolysis & putrefaction (bacteria
attack).
3. To maintain the shape & volume of tissue for
subsequence processes.
4. To prepare tissue & leave it in a condition which
allow clear staining of sections.
• Fixation should be carried out as soon as possible
after removal of the tissues (in the case of surgical 23

pathology) or soon after death (with autopsy).

Classification of histological fixative
• Histological fixatives are those fixatives which
are used for the purpose of preserving general
morphology and inclusion of the tissue.
Classification of can be based on either of the
following:
1. According to their mechanism of action
2. According to their constituents
3. According to reaction with soluble proteins
1. According to their constituent:
A. Simple fixatives(individual)
B. Compound fixatives(two or more)
• Micro-anatomical fixatives
• Cytological fixatives
2. According to mechanism of action:
• Aldehydes
• Mercurial's
• Alcohols
• Oxidizing agents
• Picrates
3. According to their reaction with soluble
proteins.
• Coagulant
• Non-coagulant
 Classification of fixatives

• According to their constituent

• Fixatives can be classified as;
• Simple fixatives and
• Compound fixatives

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 Simple fixatives

• Consists of one chemical substance in solution.

Examples are;
• Formaldehyde,
• Mercuric chloride,
• Potassium dichromate,
• Chromic acid,
• Osmium tetroxide,
• Picric acid,
• Acetone, 29

• Trichloroacetic acid.
 Compound fixatives

• Are made of two or more fixing agents.

• They are divided into;
A. Micro-anatomical fixatives
B. Cytological fixatives
i. Nuclear fixative
ii. Cytoplasmic fixative

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 Micro-anatomical fixative

• These preserve the micro-anatomical structure of

the tissue with correct relationship of tissue layers
and cells .
• Micro anatomical fixatives commonly used;
• Formal sublimate,
• Zenker’s fluid,
• Helly’s fluid,
• Bouin’s fluid,
• Heidenhain’s ‘Susa’, 31

• Buffered 10% formalin pH 7.0

 Cytological fixative

• These generally preserve the intracellular

structures and inclusions of the cell.
• Nuclear fixative
• These preserve the nuclei and its inclusions.
• The common nuclear fixatives include;
• Carnoy’s fluid,
• Flemming’s fluid and
• Clarke’s fluid
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 Cytological fixative…

II. Cytoplasmic fixatives

• These preserve the cell cytoplasm and
cytoplasmic inclusions.
• Common cytoplasmic fixatives are;
• Muller’s fluid,
• Orth’s fluid and
• Schaudinn’s fluid

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 Classification of fixatives cont..

• According to mechanism of action

• There are five major groups of fixatives,
classified according to mechanism of action:
• Aldehydes
• Mercurials
• Alcohols
• Oxidizing agents
• Picrates
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 Aldehydes

• Aldehydes include formaldehyde (formalin) and

glutaraldehyde.
• Tissue is fixed by cross-linkages formed in the
proteins, particularly between lysine residues.
• This cross-linkage does not harm the structure of
proteins greatly, so that antigenicity is not lost.
• Therefore, formaldehyde is good for
immunoperoxidase techniques.
• Formalin penetrates tissue well, but is relatively slow. 35
 Aldehydes…

• The standard solution is 10% neutral buffered formalin.

• A buffer prevents acidity that would promote autolysis and
cause precipitation of formol-heme pigment in the tissues.
• Glutaraldehyde causes deformation of alpha-helix structure
in proteins so is not good for immunoperoxidase staining.
• However, it fixes very quickly so is good for electron
microscopy.
• It penetrates very poorly, but gives best overall cytoplasmic
and nuclear detail.
• The standard solution is a 2% buffered glutaraldehyde
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 Mercurials

• Mercurials fix tissue by an unknown mechanism.

• They contain mercuric chloride and include such
well-known fixatives as B-5 and Zenker's.
• These fixatives penetrate relatively poorly and
cause some tissue hardness, but are fast and give
excellent nuclear detail.
• Their best application is for fixation of hematopoietic
and reticuloendothelial tissues.
• Since they contain mercury, they must be disposed 37
of carefully.
 Alcohols

• Alcohols, including methyl alcohol (methanol) and

ethyl alcohol (ethanol), are protein denaturants and
are not used routinely for tissues because they
cause too much brittleness and hardness.
• However, they are very good for cytologic smears
because they act quickly and give good nuclear
detail.
• Spray cans of alcohol fixatives are marketed to
physicians doing PAP smears, but cheap
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hairsprays do just as well.
 Oxidizing agents

• Oxidizing agents include permanganate

fixatives (potassium permanganate), dichromate
fixatives (potassium dichromate), and osmium
tetroxide.
• They cross-link proteins, but cause extensive
denaturation.
• Some of them have specialized applications, but
are used very infrequently
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 Picrates

• Picrates include fixatives with picric acid.

• Foremost among these is Bouin's solution.
• It has an unknown mechanism of action.
• It does almost as well as mercurials with nuclear
detail but does not cause as much hardness.
• Picric acid is an explosion hazard in dry form.
As a solution, it stains everything it touches
yellow, including skin.
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 Fixation artifacts

• These are artificial pigments produced in the

tissue during histological techniques fixation.
Some common artifacts are;
a. Formalin pigment
b. Mercuric chloride pigment
c. Chrome deposits
d. Pink disease artifacts

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 Factors affecting fixation

• There are a number of factors e.g.

• Buffering
• Penetration
• Volume
• Temperature
• Concentration
• Time interval

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 Key Points
• Fixation Is the preservation of biological tissues from
decay due to autolysis and putrefaction.
• Fixation should be carried out as soon as possible after
removal of the tissues (in the case of surgical
pathology) or soon after death (with autopsy) to prevent
autolysis.
• The choice of fixative depends on the staining
technique (histological staining, immunohistochemistry,
in situ hybridization) to be used later.
• Fixatives are classified as simple and compound 43
fixative
 Review Questions

• Define fixatives
• List classes of fixatives
• Enlist factors affecting fixation
• Explain different modes of fixation

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 References:
• F.J. Baker, R.E. Silverton, Introduction to Medical Laboratory
Technology, 7th Edition, (2001) Oxford University Press;
• F.I. Carson, Histotechnology, A Self Instructional Text, 3 rd
edition (2009) ASCP Press
• R.A.B Drurry, E.A. Wallington, Carleton’s Histological
Technique, (1976) Oxford University Press
• J.D Bancroft, M. Gamble, Theory and Practice of Histological
Techniques,6th edition,(2008) Churchill Livingstone Elsevier.
• Histology Video Links:
• Histology Lab Videos - SMPH Video Library
• https://videos.med.wisc.edu/events/140
• Videos - University of Missouri 45

• http://www.ihcworld.com/video/histology-video.htm