FIXATION AND TYPES OF FIXATIVE AGENTS

PRESENTED BY DR. SAIMA MASOOD

 FIXATION

 The broad objective of tissue fixation is to preserve cells and tissue components in a “life-like
state” and to do this in such a way as to allow for the preparation of thin stained sections. 
 The course during fixation and the steps that follow there are substantial changes to the
composition and appearance of cell and tissue components and these are quite far removed
from the ideal “life-like state.
 During fixation with care we can produce consistent chemical and physical characteristics in
tissue sections which allow patterns to be observed, morphological and chemical changes to
be noted and comparisons made. These observations allow us a view of a dynamic ever-
changing environment “fixed” at a particular point in time  and may enable a
histopathological diagnosis.
IMPORTANCE OF FIXATION:
 For practical purposes fixation aims to prevent or arrest the degenerative processes
which commence as soon as a tissue is deprived of its blood supply.
 Autolysis which results in tissue digestion by intracellular enzymes released when
organelle membranes rupture and bacterial decomposition or putrefaction which is
brought about by micro organisms which may already present in the specimen, are
processes that must be prevented.
 Loss and diffusion of soluble substances must be avoided as far as possible by
precipitation or coagulation of these components or by cross-linking them to other
insoluble structural components.
 The tissues must be largely protected against the deleterious effects of 
tissue processing including infiltration with hot wax but importantly tissues must
retain reactivity to stains and other reagents including antibodies and nucleic acid
probes.
CONTINUE…
 It is important to realise that a fixative will initially produce a number of changes to
the tissues in what is usually an aqueous environment.
 These will include shrinkage, swelling and hardening of various components. Despite
these initial effects tissues will undergo further changes during processing when they
are placed in a non-aqueous environment. 
 For example fixation in 10% buffered formalin initially causes slight swelling of
tissue specimens. During processing however the specimen may shrink 20% - 30% of
its volume. 
 The particular fixative employed will also influence the degree to which individual
elements will stain with various histochemical and immuno-histochemical
 reagents.  Thus the total effect on tissues of a particular fixative should be assessed
after a tissue has been processed, sectioned and stained to demonstrate the required
elements.
Figure 1: A paraffin section of kidney that
has been fixed using neutral buffered
formalin. This is an example of well-fixed
tissue showing good nuclear and
cytoplasmic morphology with minimal
shrinkage showing clearly defined
basement membranes and cell margins
Figure 2: A paraffin section of kidney that has been
fixed using neutral buffered formalin. This is an
example of poorly-fixed tissue showing inferior
nuclear and cytoplasmic morphology with excessive
shrinkage and poorly defined cell margins. Note the
vacuolation and fragmentation of both nucleus and
cytoplasm of cells of the distal tubule and retraction
of the glomerulus due to shrinkage.
TYPES OF FIXATION:
 Fixation of tissues can be achieved by chemical or physical means.
 Physical methods include heating, micro-waving and cryo-preservation (freeze
drying). Heat fixation is rarely used on tissue specimens, its application being
confined to smears of micro organisms. However, microwave fixation, which can be
regarded as a form of heat fixation, is now widely practiced in routine laboratories.
 Cryo-preservation usually in the form of freeze drying has some applications in
histochemistry but is not usually applied to diagnostic tissue specimens.
 Chemical fixation is usually achieved by immersing the specimen in the fixative
(immersion fixation) or in the case of small animals or some whole organs such as a
lung by perfusing the vascular system with fixative (perfusion fixation).
 For some specialised histochemical procedures fixatives have occasionally been
applied in the vapour form. For example paraformaldehyde and osmium tetroxide can
be used to vapour-fix freeze-dried tissues.
• Fixative solutions may contain a single fixative agent dissolved in a solvent such as
water or alcohol or more commonly, a buffer solution to stabilize pH.
• Some popular fixative solutions contain several different fixing agents in combination
the rationale being that the defects in one agent can be compensated for by the
addition of another. For example acetic acid is present in some formulations to
counter the shrinkage caused by other agents such as ethanol. 
THEORETICAL BASIS OF FIXATION:
 Fixation may be considered a complex series of chemical events. Although we can
now define some of these “events” our understanding of much of what happens
during fixation is still incomplete.
 Cells and extracellular components contain peptides and proteins, lipids and
phospholipids (membranes), carbohydrates and carbohydrate complexes various
types of RNA and DNA and so on.
 How these elements will react during fixation will depend on the type of fixation, the
fixing agent used and the fixation conditions. Some tissue elements will chemically
react with the fixative be stabilized by cross-linking and thus preserved others may be
unaffected by the fixative but trapped within a cell or tissue by other fixed elements
CLASSIFICATION OF FIXING AGENTS AND THE
MECHANISMS OF FIXATION
 Traditionally fixing agents were termed “coagulant” or “non-coagulant” based on
their effect on soluble proteins in solution. 
 Coagulant fixatives: were said to result in a permeable meshwork of protein strands
 Non-coagulant fixatives which are additive in nature formed extensive cross-links
producing a less permeable gel. These terms are still encountered in modern
histological literature but a more systematic approach has recently been taken to
classification. 
 There are two major mechanisms which are important in fixation of proteins and
protein complexes: denaturation, and addition and cross-link formation.
 MECHANISM…
 Denaturation: Most commonly this effect is induced by dehydrants such as the
alcohols or acetone. These reagents remove and replace free water in cells and tissues
and cause a change in the tertiary structure of proteins by destabilizing hydrophobic
bonding.
 Hydrophobic areas, frequently found on the inside of protein molecules, are released
from the repulsion of water and become free to occupy a greater area.
 In hydrophilic areas of protein water molecules are loosely bound by hydrogen bonds
and removal of water also destabilizes these bonds.
 The changes produced in the conformation of the protein molecules cause a change in
the solubility of the protein rendering water soluble proteins insoluble, a change that
is largely irreversible if the protein is returned to an aqueous environment.
 CONTINUE…
 Addition and cross-link formation: The non-coagulant fixing agents chemically react
with proteins and other cell and tissue components becoming bound to them by
addition and forming inter-molecular and intra-molecular cross-links. Because these
agents are reactive compounds they bind to a variety of chemical groups in tissues,
often affecting the charge at the site of attachment.
 This can have an effect on the subsequent staining characteristics of a particular
protein as well as altering its molecular conformation and thus its solubility. For
example, tissue fixed with formaldehyde stains poorly with eosin because
formaldehyde reacts extensively with amino groups to form methylene bridges and
thus these groups are no longer available to bind negatively charged dye molecules
such as those of eosin.
 CONTINUE….
 The extent to which additive fixatives form cross-links varies considerably. For example
glutaraldehyde is more effective at forming cross-links than formaldehyde. 
 This explains why it so effectively preserves the ultrastructure of cells and is the fixative
of choice for electron microscopy. It also explains why glutaraldehyde-fixed tissues
stain poorly with conventional dye-staining methods.
 The chemical reactions of tissue fixation are quite well understood in the case of some
agents such as formaldehyde but our knowledge of the mechanisms involved with some
other agents is incomplete.
 Antigen-retrieval methods:  in immunohistochemistry have shown that some of the
reactions of fixation are reversible, particularly those of formaldehyde but there is
considerable variation in the quality of antigen preservation with various agents.
 The preservation of antigenicity has become a very important consideration when
choosing a fixative.
Figure 3: A paraffin section of the mucosa of
small intestine that has been fixed in neutral
buffered formalin, a cross-linking fixative.
Nuclear and cytoplasmic preservation is
satisfactory but some cellular shrinkage is
present
Figure 4: A paraffin section from the mucosa of
small intestine that has been fixed in 95%
ethanol, a denaturing fixative. While nuclear
preservation is fair there is substantial shrinkage
of cytoplasmic and extracellular elements.
Compare the morphology demonstrated here
with that shown in Figure 3, which was
photographed at the same magnification.
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