CHAPTER FIVE
PRESERVATION OF
HISTOPATHOLOGICAL
SPECIMEN(By Mitiku Solomon)
06/24/2024 By Mitiku Solomon 1
Learning objectives
• Upon completion of this chapter, the student
will be able to:
• Define fixation and fixatives.
• Describe specimen preserved by fixation
• Explain ways of classifying fixatives.
• Discuss factors that affect quality of fixation.
• Describe agonal changes and artifacts.
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FIXATION
Definition :It is a complex series of chemical events
which brings about changes in the various chemical
constituents of cell like hardening, however the cell
morphology and structural detail is preserved.
Unless a tissue is fixed soon after the removal from
the body it will:-
undergo degenerative changes due to autolysis and
putrefaction(The breakdown of tissue by bacterial
action often with formation of gas) so that the
morphology of the individual cell will be lost.
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FIXATION …
• Histochemical fixatives:-For a most of the
histochemical methods, It is best to use
[Link] are rapidly frozen or freeze
dried. Usually such sections are used unfixed
but if delay is inevitable then vapour fixatives
are used([Link] vapour is obtained
by heating paraformaldhyde at temp. b/n
50℃and 80℃.Blocks of tissue require 3-
[Link] require 30min to 1hr.
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Fixation of specific substance
[Link]
• The retention of glycogen is thought to be the
result of trapping in a matrix or mesh of fixed
protein, or due to its covalent binding to
protein, which renders it insoluble in water.
The use of alcohols has been the main method
of fixing glycogen in tissues.
Earlier fixatives included ice-cold picro-alcohol-
formalin or cold alcohol.
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Cont..
Chmical assays on rat liver have shown 100%
ethanol to be clearly superior for the fixation of
glycogen.
Bouin's fixative is also a useful fixative for glycogen.
[Link]
With standard methods of fixation, lipids are largely
lost from tissues during processing and only two
reagents fix lipids in the true sense of rendering them
insoluble.
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Cont..
• These are osmium tetroxide and chromic acid, both
of which alter the chemical reactivity of the lipid
considerably.
• Various additives have been mixed with
glutaraldehyde in order to demonstrate lipids in
electron microscopy.
• 3. Proteins
• The fixation of tissue proteins by aldehydes is
largely through production of cross-linkages
between various reactive groups in proteins.
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Cont..
4 Mucosubstances
• The loss of mucosubstances from tissue during
fixation is well recognized and many fixatives
have been suggested to prevent.
.Formalin has always been an essential
component of whatever fixative used to ensure
the preservation of proteoglycans.
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Cont..
• An appreciable proportion of tissue hetero-
and proteoglycans remains soluble unless
subject to further precipitation in 70-80%
ethanol (for 3-6 days) before clearing and
embedding in paraffin.
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Cont..
• The most successful method for preserving all
types of mucin is freeze drying followed by hot
formaldehyde vapor with the all necessary
safety precautions.
5. Nucleic acids and nucleoproteins
• The nucleic acids exist in many different states
of polymerization and any method of fixation
induces changes in their physical state
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Cont..
• Formalin is not a particularly good fixative for
nucleic acids and nucleoproteins as it blocks a
large number of reactive groups reducing their
subsequent staining by both basic and acid
dyes
This can be improved by adding mercury or
chromium salts.
• Precipitant fixatives like alcohol, acetic acid,
and Carnoy's fluid are preferable.
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Cont..
• these agents precipitate nuclear proteins and at the
same time progressively break the bonds between
nucleic acids and proteins, thereby increasing the
number of acid groups available for staining.
6 Enzymes
• Enzyme activity is best demonstrated
histochemically in fresh frozen sections.
• The most common methods of preserving enzymes
for paraffin embedding are fixation in alcohol or
acetone, usually at 4°C.
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Agonal changes and artifacts
• as most tissues are removed surgically, the
following agonal changes may occur.
• Organ or tissue become anoxic when tissue placed
on fixatives;
Anoxic changes are noticed;
Enzymes such as those concerned with oxidative
phosphorylation are lost within 10 minutes;
There will be tissue variation;
Some degree of autolysis will also occur.
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Cont..
• Artifacts caused by fixatives include:
• Both large and small molecules diffuse from the tissue in
to fixation solution;
• Chemical changes caused by fixation may give false
histochemical reactions in tissue;
• While removing excess mercuric chloride after fixation,
substances such as histidine, tyrosine, may also be
removed
• Using substances specific fixatives results loss of many
other cellular substances.
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Cont..
• Formaldehyde at pH 7.0 causes the loss of
about 60% of catecholamines from chromaffin
granules.
• Formalin is an inadequate fixative for the
proteoglycans of many pituitary glands and
many neuropeptides such as luteinizing
hormone-releasing hormones are ethanol
soluble and may be lost during dehydration.
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Cont..
• Enzymes may be released during fixation and
ions may be lost from tissues.
• Prolonged fixation in formaldehyde results in
the loss of water-soluble materials particularly
when fixation exceeds 6 hours.
• Proteins are degraded by osmium tetroxide.
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Cont…
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Review questions
1. What are fixation and fixatives?
2. Explain the characteristics of a good fixative.
3. Discuss factors affecting quality of fixation.
4. Discuss about agonal changes and artifacts.
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References
1. Sir William Lyon’s Road,John Crocker
the science of laboratory diagnosis, second edition
University of Warwick Science Park, Department of
Cellular Pathology,
2. Berhanu Seyum, Jemal Yimam Haramaya
university ,Histopathology for Medical Laboratory
Technology students in Ethiopia ,Lecture note
series in collaboration with The Carter Center ,2006
Addis Ababa.
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