FIXATION OF TISSUES

By
ISAAC ASIMADU MENSAH
 Introduction
• A fixative is a substance which preserves after death/ or
removal, the shape, structure, relationship and chemical
constituents of tissues and cells.
• It is mainly due to the action of fixatives on the protein
elements of cells and tissues that the structural stabilization is
achieved.
• The fixed tissue resembles as close as possible to the form
which it had during life.
• The fixative acts by minimizing the loss or enzymatic
destruction of cellular and extracellular molecules,
maintaining macromolecular structures and protecting tissues
from destruction by microorganisms.

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 Intro cont
• Appropriate fixation of tissues for histological examination is
extremely important. Without attention to this process, the range of
tests performed in a modern histopathology laboratory will be
rendered ineffective and practically useless.
• The concept of fixation of biological tissues in order to understand
biological function and structure has led to the development of
many types of fixatives.
• The foundation of all good histological preparations is adequate and
complete fixation. Faults in fixation cannot be remedied at any later
stage, and the finished section can only be as good as its primary
fixation.
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 Intro cont

• The mechanisms and principles by which specific fixatives act to

harden and preserve tissues and prevent the loss of specific molecules
fall into broad categories:
 Covalent addition of reactive groups and of cross links
 Dehydration and effects of acids

 Salt formation and heat

 Combination of any of the above

• Although each fixative has advantages, they all have many

disadvantages :
 Molecular loss from ‘fixed’ tissues
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 Intro cont

 Swelling or shrinkage of tissues during the process,

 Variations in the quality of histochemical and
immunohistochemical staining.
 The Ability to perform biochemical analysis accurately,
 Varying capabilities to maintain the structures of cellular
organelles.
 No ideal fixative
 Fixatives are selected based on their ability to produce a final
product needed to demonstrate a specific feature of a specific
tissue.
 10% neutral buffered formalin

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 Qualities of an ideal fixative

 Support high quality and consistent staining with H&E, both

initially and after storage of the paraffin blocks for at least a
decade.
 The ability to prevent short- and long-term destruction of the
micro-architecture of the tissue by stopping the activity of
catabolic enzymes and hence autolysis,
 Minimizing the diffusion of soluble molecules from their
original locations.
 Inanimation of infectious agents, which helps maintain tissue
and cellular integrity (putrefaction).

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 Qualities of an ideal fixative cont

 Good toxicological and flammability profiles that permit the

safe use of the fixative.
 Permit the recovery of macromolecules including proteins,
mRNA, and DNA without extensive biochemical modifications
from fixed and paraffin-embedded tissues.
 Useful for a wide variety of tissues, including fatty, lymphoid,
and neural tissues.
 Should preserve small and large specimens and support
histochemical, immunohistochemical, in situ hybridization and
other specialized procedures.
 Penetrate and fix tissues rapidly

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 Qualities of an ideal fixative cont

 Shelf life of at least one year.

 Compatible with modern automated tissue processors. The
fixative
 Readily disposable or recyclable.
 Support long-term tissue storage giving excellent microtomy
of paraffin blocks.
 Should be cost-effective.

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 Types of fixation

• Fixation of tissues can be accomplished by 2 methods:

 Phyysical
 Chemical
 Physical methods such as heating, microwaving, and freeze-drying
are not used commonly in routine medical pathology anatomy,
and histology, except for the use of dry heat fixation of
microorganisms prior to Gram staining.
 Heat fixation
• The simplest form of fixation.
• Picking up a frozen section on a warm microscope slide both
attaches the section to the slide and partially fixes it by heat and
dehydration.

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 Types of fixation cont

• Adequate morphology could be obtained by boiling tissue in

normal saline, however, in histopathology heat is primarily
used to accelerate other forms of fixation as well as the steps
of tissue processing.
 Microwave fixation
• Microwave heating speeds fixation and can reduce times for
fixation of some gross specimens and histological sections
from more than 12 hours to less than 20 minutes.
• Microwaving tissue in formalin results in the production of
large amounts of dangerous vapors.
o Should not be done in the absence of a hood for fixation of
microwave processing system designed to handle these vapors
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 Types of fixation cont

 Freeze drying and freeze substitution

• Useful technique for studying soluble materials and small
molecules.
• Tissues are cut into thin sections, immersed in liquid nitrogen,
and the water is removed in a vacuum chamber at −40°C.
• The tissue can be post-fixed with formaldehyde vapor.
• In substitution, specimens are immersed in fixatives at −40°C,
such as acetone or alcohol, which slowly remove water
through dissolution of ice crystals, and the proteins are not
denatured; bringing the temperature gradually to 4°C will
complete the fixation process
• Primarily used in the research.

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 Types of fixation cont

 Chemical fixation utilizes organic or non-organic solutions to

maintain adequate morphological preservation.
• Chemical fixatives can be considered as members of three
major categories: coagulant, crosslinking, and compound
fixatives.
 Coagulant fixatives
• Both organic and non-organic solutions may coagulate
proteins, making them insoluble.
• Cellular architecture is maintained primarily by lipoproteins
and by fibrous proteins such as collagen; coagulating such
proteins maintains tissue histomorphology at the light
microscopic level.

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 Types of fixation cont
• Coagulant fixatives result in cytoplasmic flocculation as well as
poor preservation of mitochondria and secretory granules,
hence such fixatives are not useful in ultrastructural analysis.
 Dehydrant coagulant fixative
• The most commonly used coagulating fixatives are alcohols
(e.g. ethanol, methanol) and acetone.
• Molecules of water participate in hydrogen bonding in
hydrophilic areas of proteins; so removal of water destabilizes
this hydrogen bonding. This disrupt the tertiary structure of
proteins.
• Removal of water may also partially reverse the structure of
proteins.

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 Types of fixation cont
• Once the tertiary structure of a soluble protein has been modified,
the rate of reversal to a more ordered soluble state is slow.
• Most proteins after coagulation remain insoluble even if returned to
an aqueous environment.
• Disruption of the tertiary structure of proteins, i.e. denaturation,
changes their physical properties, potentially causing insolubility and
loss of function.
• Denaturing of proteins by alcohol will depend on the choice and
concentration of alcohol, the presence of organic and non-organic
substances, and the pH and temperature of fixation.
•
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 Types of fixation cont

 Other types of coagulant fixative

• Acidic coagulants such as picric acid and trichloroacetic acid
change the charges on the ionizable side of proteins and
disrupt electrostatic and hydrogen bonding. Eg
(–NH2 → NH3+) and (COO− → COOH).
• These acids also may insert a lipophilic anion into a
hydrophilic region and hence disrupt the tertiary structures of
proteins.
• Acetic acid coagulates nucleic acids but does not fix or
precipitate proteins; it is therefore added to other fixatives to
prevent the loss of nucleic acids.

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 Types of fixation cont

• Trichloroacetic acid (Cl3CCOOH) can penetrate hydrophobic

domains of proteins and the anion produced(–C–COO−) reacts
with charged amine groups. This interaction precipitates
proteins and extracts nucleic acids.
• Picric acid or trinitrophenol slightly dissolves in water to form
a weak acid solution (pH2.0). In reactions, it forms salts with
basic groups of proteins, causing the proteins to coagulate.
 Non coagulant cross linking fixatives
• These chemicals form cross-links within and between proteins
and nucleic acids as well as between nucleic acids and
proteins.
• Covalent additives fixatives
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 Types of fixation cont
• Examples include formaldehyde, glutaraldehyde, and other
aldehydes, e.g. chloral hydrate , metal salts such as mercuric
and zinc chloride, and other metallic compounds such as
osmium tetroxide.
o Formaldehyde fixation
• Formaldehyde in its 10% neutral buffered form (NBF) is the
most common fixative used.
• In an aqueous solution formaldehyde forms methylene
hydrate, a methylene glycol as the first step in fixation.
H2C=O + H2O →HOCH2 OH
• Methylene hydrate reacts with several side chains of proteins
to form reactive hydroxymethyl side groups (-CH2-OH)

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 Types of fixation cont

• Formaldehyde also reacts with nuclear proteins and nucleic

acids.
• It penetrates between nucleic acids and proteins and
stabilizes the nucleic acid-protein shell, and it also modifies
nucleotides by reacting with free amino groups, as it does
with proteins.
• Formaldehyde reacts with C=C and –SH bonds in unsaturated
lipids, but does not interact with carbohydrates.
• The side chains of peptides or proteins that are most reactive
with methylene hydrate, and hence have the highest affinity
for formaldehyde, include lysine, cysteine, histidine, arginine,
tyrosine, and reactive hydroxyl groups of serine and threonine
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 Types of fixation cont
o Osmium tetroxide fixation
• (OsO4) is a toxic solid, soluble in water as well as non-polar
solvents.
• Reacts with hydrophilic and hydrophobic sites including the
side chains of proteins, potentially causing crosslinking.
• The reactive sites include sulfydryl, disulfide, phenolic,
hydroxyl, carboxyl, amide, and heterocyclic groups.
• Secondary fixative for electron microscopy examination
• Staining of lipids in frozen sections
• OsO4 fixation causes tissue swelling but this is reversed during
dehydartion steps
• Calcium /Sodium chloride

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 Types of fixation cont
 Dehydrant fixatives
• Remove free and bound water, causing a change to the
tertiary structure of proteins so that they precipitate, leaving
the nucleic acids relatively unchanged.
• Extraction of lipids
• May cause excessive shrinking of tissue components after
more than 3-4 hours of fixation.
• Ethanol, methanol, acetone, Clarke’s solution, Carnoy’s.
 Dehydrant cross linking fixatives
• These are compound fixatives with both dehydrant and
crosslinking actions. They include alcohol-formalin mixtures.
• Eg: Rossman’s solution

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 Factors affecting the quality of fixation

• Buffers and Ph
 Phosphate, cacodylate, bicarbonate, Tris, and acetate
• Duration of fixation and size of specimen

• Temperature of fixation
• Concentration of fixative
• Osmolality of fixative and ionic composition

• Nature of specimen
• Agitation

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 Effects of tissue fixation

 Confers chemical stability to the tissue

 Hardens the tissue (helps in further handling)

 Halts enzyme autolysis
 Halts bacteria putrefaction

 May enhance later staining techniques

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 Compound fixatives
 Mercuric fixative

• A problem with fixation in mercury solutions is that several

types of pigment may combine with the mercury.
• These pigments are removed from sections by using iodine
treatment followed by sodium thiosulfate.
 Zenker’s solution - good fixative for bloody specimens and
trichrome stains.
 B 5 - for bone marrow, lymph nodes, spleen, and other
hematopoietic tissues.

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 Compound fixatives con’t

 Picric acid fixatives

• The picric acid may impart the yellow colour to the tissue.

• Can be removed by 70% ethanol, lithium carbonate, or another

acid dye, separately or during the staining sequence.
 Bouins’ solution - Excellent general fixative for connective tissue
stains
 Hollande’s solution - A useful fixative for gastrointestinal biopsies
and endocrine tissue;

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 Neutral buffered 10% formalin

• Tap water …………………………………………….900 ml

• Formalin (37% formaldehyde solution) .....................100 ml
• Sodium phosphate, monobasic, monohydrate………..4 g
• Sodium phosphate, dibasic, anhydrous ……………. 6.5 g

• The pH should be 7.2–7.4

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 N S
T I O
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Q U
N Y
A
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