HISTOLOGY

LECTURE # 12
INTRODUCTION TO TISSUE FIXATION
Rationale:
Fixation is the most essential part in histology. Here is where everything starts. A
well fixed tissue is the key for a good slide and therefore a good interpretation for diagnosis.
Here we will learn the several types of fixation available their advantages and disadvantages.
Objective:
Once completed this lecture, the student should be able to:
a)Describe the various fixatives and their uses.
b)Learn the difference between autolysis and putrefaction
c)Differentiate the fixatives that could impact the final results.
d)Learn the chemicals and reagents used in each fixative.
TISSUE FIXATIVE
Introduction
Fixation -is the most important step through the process of histology. The purpose of the
fixative is to stabilize the protein in the tissue. Once the tissue is removed from the body
it will go through a process of self-destruction. This process is known as Autolysis which
starts soon after the cell death creating an enzyme attack, which in place causes the
breakdown of protein and eventual liquefaction of the cell.
Autolysis is more severe in tissues which are rich in enzymes, such as the liver, brain and
kidney, and is less rapid in tissues such as elastic fibers and collagen. By light microscopy,
autolyzed tissue presents a `washed-out' appearance with swelling of cytoplasm, eventually
converting to a granular, homogeneous mass which fails to take up stains. If tissue is left
without any preservation, then a bacterial attack will occur, this process is known as
Putrefaction.-The objective of fixation is to preserve cells and tissue constituents in as close a life-like
state as possible and to allow them to undergo further preparative procedures without
change. Fixation arrests autolysis and bacterial decomposition and stabilizes the cellular and
tissue constituents so that they withstand the subsequent stages of tissue processing.
Fixation should also provide for the preservation of tissue substances and proteins,
therefore, it is the first step and the foundation in a sequence of events that culminates in
the final examination of a tissue section.
FUNCTION OF FIXATIVES
A. Help Maintain a proper relationship between cells and extracellular substances:

Connective Tissue Fibers: Collagen, Reticulin, Elastin

Amorphous ground substances

B. Brings out differences in refractive indexes and increases the visibility or
contrast between different tissue elements.
R.I. = Velocity of light in Air/Velocity of light in a liquid or solid medium
C. Render cell constituents insoluble, with tissue proteins serving as the primary
target for stabilization.
ACTION OF FIXATIVES
A. Methods of Stabilizing Proteins
1.Heat (Physical method) this method is becoming more used in the histology
laboratories with the introduction of microwave fixation.
2. Desiccation (Physical method) Rarely used in histology. Touch preparation s
for Giemsa stains are probably the most common used for this method.
3. The use of one or more chemical reagents (Chemical method) this is
considered the primary method of fixation. These chemical reagents may be
classified as additive and non-additive or coagulant and non-coagulant.
Additive they chemically link or bind to the tissue and change it.
Non-additive include organic compounds such as acetone and alcohols,
which act on the tissue without chemically combining with the tissue.
Ex: Methyl or Ethyl Alcohols
B. Coagulant & Non-Coagulant
1.Coagulation will allow the solutions to penetrate into the interior of the
tissue very easily.
2.Non-coagulant-they act by creating a gel barrier that makes solutions
more difficult to penetrate to the interior of the tissue.
To further understand this concept of coagulant and non-coagulant, take a look to the
following examples:
(A) Coagulant
(B) Non-Coagulant
Imagine that we have a piece of tissue in the middle of both examples. Which will you think
that the solutions will penetrate easily to the tissue? Answer: (A) Coagulant: these types of
fixative will render the tissue so that it will accept subsequent solution very easily.
If we look at (B) Non-coagulant, theses types of fixative will make it difficult for subsequent
solutions to penetrate.
Coagulant Fixatives Reagents includes:
Alcohol
Zinc salts
Mercuric chloride
Chromium trioxide
Picric Acid
Non-Coagulant Fixatives Reagents includes:
Formaldehyde
Gluteraldehyde
Osmium Tetroxide
Potassium Dichromate
Acetic Acid
FACTORS AFFECTING FIXATION
Temperature
The factor influenced by temperature is the morphology of the tissue. Normally the fixation
of specimens for standard histology is carried out at room temperature for convenience. For
electron microscopy and some histochemical procedures, the temperature for fixation is
usually 0-4C. The use of chemical reactions at higher temperatures to penetrate tissue,
including those involved in the fixation process will increase the rate of penetration of the
fixative to the specimen, however, an increase in temperature will also increase the rate of
autolysis and diffusion of cellular components.
Size
The penetration of fixatives into tissue is a relatively slow process and tissue blocks should
either be small or thin, in order to obtain satisfactory fixation. Large specimens should be
opened and washed of contents or sliced thinly before placement in fixative. For routine
processing the recommended thickness of the specimens should be no more than 3mm
thick (not thicker than a nickel). For shorter processing schedules the tissue should be
of thinner sections, Ex: Kidney, liver, heart biopsies)
Volume Ratio
The recommended ratio of the tissue volume to the fixative volume is at least 15 to 20
times greater than the tissue volume. If a specimen receives more than the recommended
volume the effect is none, we have plenty of fixative to the tissue volume. However, if the
volume of the fixative is less than the volume of the tissue, we can see some problems such
as under fixed tissue (poor fixation).
Time
It is common practice to fix 2 mm thick tissue blocks in buffered formalin for 4-8 hours,
possibly followed by a period in formol sublimate. Large specimens and viscera are cut into
5 mm slices or viscera are emptied and pinned out on a board, before fixing overnight in
buffered formalin. This allows easier handling, examination and dissection, particularly for
the sampling of lymph nodes. In the case of electron microscopy, diced tissues are fixed for
3 hours in glutaraldehyde before placing in a holding buffer such as sodium cacodylate.
There is evidence that prolonged fixation in aldehydes can cause shrinkage and hardening of
tissue and severe inhibition of enzyme activity. Prolonged fixation with oxidizing fixatives
can degrade tissues by oxidative cleavage of proteins and loss of peptides. The tissue
should be placed in fixative immediately after surgery, as well as autopsies should be
performed immediately after death. The more time it elapses between interruption of the
blood supply and fixation, the more post-mortem changes will be observed under the
microscope.
Small intestine well preserved
Autolyzed Small intestine
Carson Book, Page 5, Image 1-2
Notice how is missing the epithelium
Choice of Fixative
The method of fixation should be selected immediately once the specimen is presented.
The fixative should be selected with caution avoiding fixatives that may affect future
histochemical studies. For example tissue that will be used for Immunofluorescence studies
should be preserved in a transport media. If future studies include a diagnostic for Gout
(Gophus Typhus), a fixative containing water will not work since the water will dissolve the
Urate crystals which are essential in the study, so the fixative of choice is absolute alcohol.
Enzyme studies are also very peculiar when it comes to fixatives, frozen sections are
preferred for these methods.
Electron Microscopy the choice is Gluteraldehyde. Post-fixation which is also considered a
mordanting process will help with the enhancement of the staining by allowing a better
penetration of solution to the tissue structures to be observed. Example: Masson Trichrome.
Penetration
Fixatives penetrate the tissue at different rates. These rates can be affected by heat. The
tissue is fixed starting at the periphery of the tissue and working inward toward the center
of the tissue.
Tissue Storage
Storing wet tissue is very important because often the tissue is needed for further studies.
If the tissue has not been fixed and stored properly further studies are impossible. Tissue
fixed in Neutral buffered Formalin are usually safe to use for further studies since they can
remain in this fixative indefinitely, but this is not true for other fixatives. Non fix tissue may
remain in 70% methyl alcohol.
pH
The hydrogen ion concentration varies between fixatives, but in general, the pH should be
kept in the physiological range, between pH 4-9. If formalin is allowed to fall to a lower pH
this can produce formalin pigments. Even though in routine histology the pH is not usually
critical, in electron microscopy it is very important. The pH for the ultrastructural
preservation of great specimen the fixative should be buffered between 7.2 to 7.4.
Osmolality
The addition of a buffer to the fixative solution may alter the osmotic pressure exerted by
the solution. Hypertonic solutions give rise to cell shrinkage whereas isotonic and hypotonic
fixatives result in cell swelling and poor fixation. With electron microscopy, the best results
are obtained using slightly hypertonic solutions (isotonic solutions being 340 mOsm)
adjusted using sucrose.
REACTIONS OF THE CELL WITH FIXATIVES
Glycogen
A variety of glycogens occurs naturally and show different degrees of polymerization. The
less highly polymerized forms are not well fixed by routine fixatives and diffuse into the
fixing fluid. This occurs in cases of glycogen storage disease where glycogen is
predominantly of the lighter type. In contrast, the larger molecules of more highly
polymerized glycogens are retained with a wide variety of fixatives as well as alcohol-
containing reagents. 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. However, there appears to be stronger support for the concept that
removal, by dehydration, of bound water molecules from normal forms of tissue glycogen
decreases solubility, amounting to denaturation.
Lipids
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. These are osmium
tetroxide and chromic acid, both of which alter the chemical reactivity of the lipid
considerably. While several fixatives will preserve lipids, they generally do not alter their
solubility in the lipid solvents used in tissue processing. Baker's fixative, designed for the
preservation of phospholipids, uses formalin together with calcium and cadmium chlorides
(the last, being expensive, has subsequently been replaced by cobalt nitrate). While
phospholipids are preserved, they are not prevented from diffusing into the fixing fluid and
are still removable by fat solvents. Lipids can be demonstrated in cryostat sections fixed
with reagents containing mercuric chloride and potassium dichromate such as Elftman's
fluid, with fixation for unsaturated lipids completed over 3 days at room temperature.
Various additives have been mixed with glutaraldehyde in order to demonstrate lipids in
electron microscopy. Digitonin added to glutaraldehyde preserves cholesterol and Malachite
Green included with glutaraldehyde or Karnovsky's fixative retains various lipids such as
phospholipids, fatty acids, glycolipids and choline plasmalogen. Imidazole introduced into
the post osmication of glutaraldehyde-fixed tissue demonstrates unsaturated fatty acids and
phospholipids as electron-dense deposits.
Proteins
The fixation of tissue proteins by aldehydes is largely through production of cross-linkages
between various reactive groups in proteins. Most fixatives preserve proteins adequately in
1 to 2 days. Glutaraldehyde fixes proteins very rapidly whereas formaldehyde reacts
reversibly over the first 24 hours. Osmium tetroxide reacts with proteins by producing
cross-links and protein gels. Prolonged exposure to osmium tetroxide causes the breakdown
of proteins.
Mucosubstances
Among the mucosubstances are the single component polysaccharides such as glucose,
starch and cellulose which are referred to as homoglycans whereas those with two or more
monosaccharide components are the heteroglycans. The latter are composed of the
glycosaminoglycans such as keratosulphate and sialoglycans, and the
glycosaminoglucoronoglycans comprising hyaluronic acid, chondroitin sulphates and
heparin. Protein-polysaccharide complexes are known as proteoglycans.
The loss of mucosubstances from tissue during fixation is well recognized and many
fixatives have been suggested to prevent this. Formalin has always been an essential
component of whatever fixative used to ensure the preservation of proteoglycans, however,
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.
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. 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 since 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. However, prolonged fixation in acid fixatives
such as Carnoy's reagent profoundly alters nuclear proteins and extracts RNA and DNA.
Simple Aqueous Fixatives or Fixative Ingredients
FORMALDEHYDE
Formaldehyde, as 4% buffered formaldehyde (10% buffered formalin), is the most widely
employed universal fixative particularly for routine paraffin embedded sections. It is a gas
with a very pungent odor, soluble in water to a maximum extent of 40% by weight and is
sold as such under the name of formaldehyde (40%) or formalin (a colorless liquid).
Formaldehyde is also obtainable in a stable solid form composed of high molecular weight
polymers known as Para formaldehyde. Heated Para formaldehyde generates pure gaseous
formaldehyde which, when dissolved in water, reverts mostly to the monomer form.
Aqueous formaldehyde exists principally in the form of its monohydrate, Methylene glycol,
CH2(OH)2, and as low molecular weight polymeric hydrates or polyoxymethylene glycols. It
has been suggested that the hydrated form, Methylene glycol, is the reactive component of
formaldehyde but this has been disputed.
Four per cent formaldehyde or 10% buffered formalin is commonly prepared by adding 100
ml of 40% formaldehyde to 900 ml distilled water with 4 g sodium phosphatase, monobasic
and 6.5 g sodium phosphate, dibasic (anhydrous). To be effective, the specimen should be
completely submerged in five to twenty times its volume of fixative.
Ten per cent buffered neutral formalin preserves a wide range of tissues and has the
advantage of being a forgiving fixative. It requires a relatively short fixation time but can
also be used for long-term storage as it produces no deleterious effects on tissue
morphology with nuclear and cytoplasmic detail being adequately preserved.
Details of the fixing action of formaldehyde and other aldehydes are not known although the
general principles are understood. It is thought that the aldehydes form cross-links between
proteins, creating a gel, thus retaining cellular constituents in their in vivo relationships to
each other. Soluble proteins are fixed to structural proteins and rendered insoluble, giving
some mechanical strength to the entire structure which enables it to withstand subsequent
processing. With the aldehydes, cross-links are formed between protein molecules, the
reaction being mostly with basic amino acid lysine, although other groups such as imino,
amido, peptide, guanidyl, hydroxyl, carboxyl, SH and aromatic rings may also be involved.
Only those lysine residues which are on the exterior of the protein molecule react, these
usually accounting for 40-60% of the total lysyl residues.
Although the extent of denaturation produced by fixation this does not matter greatly in
routine tissue pathology, it is of particular importance in the detection of antigens both by
immunofluorescence and immunoenzyme techniques as well as in high resolution electron
microscopy. Similarly, the shapes of large molecules must not be changed if they are to be
recognized by biochemical analysis. Glutaraldehyde causes a loss of up to 30% of the alpha
helix structure of protein, depending on the type of protein. Fixation with osmium tetroxide
or post osmication of glutaraldehyde-fixed material causes the complete denaturation of
protein.
Formalin does not precipitate proteins and only slightly precipitates other components of the
cell. It does not harden or render albumin insoluble but subsequent hardening by alcohols is
prevented. Formalin neither preserves nor destroys adipose tissue and is a good fixative for
complex lipids but has no effect on neutral fats. Although not the fixative of choice for
carbohydrates it preserves proteins so that they hold glycogen which is otherwise readily
leeched from the cell.
Formaldehyde solution is nearly always acid. It certainly becomes acid on storage as
formalin oxidizes to formic acid, reducing its preserving capabilities such that neutralization
of the solution is a requirement. In addition, formaldehyde solution produces acid formalin
haematin pigment which can be seen in sites containing blood. If calcium carbonate is used
for neutralizing formalin the resultant solution does not retain its neutral pH and calcium
carbonate itself can deposit in tissues, leaving areas of `pseudocalcification'. Phosphate
buffers such as sodium phosphate monobasic and sodium phosphate dibasic are effective
for the neutralization of formalin and the pH of the solution produced is stable for many
months. Formalin should not be used with chromates because it readily oxidizes to formic
acid.
A concentrated solution of formalin sometimes becomes turbid on storage through the
production of Para formaldehyde which decreases the strength of the solution, but can still
be used as a fixative following filtration. Formalin favors the staining of acidic structures
(nuclei) with basic dyes and diminishes the effect of acid dyes on basic structures
(cytoplasm).
Formaldehyde is an immediate irritant to the eyes, upper respiratory tract and the skin, and
safety precautions should include proper ventilation and exhaust, limited or restricted
exposure periods and thorough washing if spilt on tissue surfaces such as the skin.
GLUTARALDEHYDE
Like formaldehyde which acts through the formation of cross-links between protein end-
groups, glutaraldehyde has also been used extensively as an agent for protein-protein
linkage and hence for fixation. An aqueous solution of glutaraldehyde (glutaric dialdehyde)
is a complex mixture at room temperature, consisting of approximately 4% free aldehyde,
16% monohydrate, 9% dihydrate and 70% hemiacetal. Free glutaraldehyde may form
polymers, or a monohydrate and a dihydrate, which may cyclize to give a hemiacetal which
in turn may also polymerize. Some favor the polymer as the reactive species while others
suggest that pure, monomer, glutaraldehyde is the best fixative and much less inhibitory to
enzymes than is the mixed monomer-polymeric product. The success of glutaraldehyde as a
cross-linking agent may also depend on the large range of different molecules present
simultaneously in the fixation solution.
When glutaraldehyde solutions are kept for long periods at ambient temperatures, there is a
tendency for precipitates to form and for aldehyde levels to fall so that some method of
purification may be required. Glutaraldehyde may be purified to the monomer form by
removing oligomers, polymers and other impurities through simple shaking with barium
carbonate, vacuum distillation or treatment with activated charcoal and chromatography on
Sephadex G-10 has produced equally good results. Vacuum distillation after prior treatment
of commercial glutaraldehyde solutions with sodium chloride and ethanol has become the
most widely used technique for purification.
There are many variations in the preparation of this fixative, including the percentage of
glutaraldehyde, additives, and buffers. Because of its low penetration, only small blocks of
tissues (1-2 mm) fix well at temperatures of 1-4C. The fixed tissue specimen can be
stored in buffer solution for many months.
The slow penetration, the requirement for cold temperature and the need for a storage
medium, have greatly limited the use of glutaraldehyde in histology. It is, however, the
most widely used fixative for standard electron microscopy.
Other uses for glutaraldehyde all of which really its cross-linking properties include the
preparation of tissue xenografts, particularly cardiac valves, chemical sterilisation and
disinfection. Glutaraldehyde has an inhibitory effect on catalase allowing the selective
demonstration of the peroxidase activity of peroxisomes.
OSMIUM TETROXIDE
The most commonly used metallic ion in fixation is osmium tetroxide which was initially a
tissue fixative used in cytology, but poor penetration limited its application in light
microscopy. It is now largely employed as a secondary fixative in electron microscopy.
Osmium tetroxide is known to form cross-links with proteins as reflected in the rapid
increase in viscosity of a protein solution when they react together, however, there is very
little additional information as to its the mechanism of action. There is some general
agreement that osmium tetroxide reacts with unsaturated lipids as it is reduced with the
formation of black compounds containing hexavalent osmium. Various hypotheses of lipid
stabilization have been postulated and these include the oxidation of double bonds between
adjacent carbon atoms to form monoesters and diesters, the binding of lipid to protein and
the conversion of unsaturated fatty acids to stable glycol osmates. More recent studies show
that the reaction of osmium tetroxide is largely with lipid rather than protein.
Osmium tetroxide is used for preservation of fine structures in electron microscopy and is
effective for small (2-3 mm) specimens. While vapors of this fixative will preserve blood
and tissue smears, its low and uneven penetration limits its application in routine light
microscopy and osmium tetroxide fixed tissues often crumble if embedded in paraffin.
Osmium tetroxide also interferes with many staining procedures.
CHROMIC ACID
Chromic acid (chromium trioxide) is a strong oxidizer that is used with other ingredients. It
has no effect on fats, penetrates slowly and leaves tissues in a state where shrinkage may
occur during subsequent processing. Chromium salts form complexes with water which
combine with reactive groups of adjacent protein chains to bring about a cross-linking effect
similar to that of formalin. The reaction of potassium dichromate with adrenal medullary
catecholamines results in the production of black or brown water-insoluble precipitates. The
dichromate-oxidation product is not only visible grossly but also in the tissue section and is
still regarded as a rapid means of identifying tissues with aromatic amines such as adrenal
medullary tumors. Potassium dichromate is never used alone and, if employed other than
for the demonstration of amines, should be washed thoroughly to remove the oxide that
forms as it cannot be removed later in processing.
Other heavy metals such as palladium chloride and uranium may result in some degree of
tissue fixation but have no practical application in histopathology.
ACETIC ACID
Acetic acid is never used alone but is often combined with other fixatives that cause
shrinkage such as ethanol and methanol. Acetic acid penetrates thoroughly and rapidly but
lyses red blood cells.
PICRIC ACID
Picric acid, when used in combination with other ingredients, leaves tissue soft and
penetrates well, precipitating all proteins. It will continue to react with the tissue structures
and cause a loss of basophilia unless the specimen is thoroughly washed following fixation.
MERCURIC CHLORIDE
Mercuric chloride (corrosive sublimate, bichloride of mercury) and other salts of mercury
were common histological fixatives in the past. These penetrate rapidly and precipitate all
proteins, reacting with a number of amino acid residues including thiol, amino, imidazole,
phosphate and hydroxyl groups. The production of hydrogen ions makes the fixative
solution more acidic and mercuric crystals deposited in the tissue need to be removed
before staining. Mercuric chloride is contained in Zenker's, Helly's, Ridley's and B5 solutions.
It should also be noted that mercuric salts are highly toxic and must not be disposed into
sewerage systems. One method of disposal is to precipitate the mercuric chloride with
thioacetamide. For example, mixing 1 liter of Zenker's solution with 20 ml of 13%
thioacetamide solution in a tightly capped container results in a precipitate of mercuric
sulphide which can be filtered out and disposed of safely.
ACETONE
Acetone is a clear, colorless, inflammable liquid which is miscible with water, ethanol and
most organic solvents. It has been used as a dehydrating agent in tissue processing and is
more volatile than alcohols and other dehydrants. It has a rapid action but causes
brittleness in tissue if exposure is prolonged and because it is volatile and inflammable,
acetone is not used in automated processing schedules. However, it has a greater solvent
action on lipids and is rapidly removed by most clearing agents, making it very useful in
manual processing procedures.
More recently, acetone has been employed as a fixative in the acetone-methylbenzoate-
xylene (AMEX) technique.This requires overnight fixation of tissues in acetone at -20C
then clearing in methylbenzoate and xylene before paraffin embedding. The product is
claimed to show better histologic preservation than is possible to obtain in frozen sections,
yet retaining reactivity for labile lymphocyte membrane antigens.
TRANSPORT SOLUTIONS
Michel Transport Medium
It is important to maintain the pH in between 7.0 to 7.2, because a lower pH can cause
variable results. This transport medium is used for specimen such as kidney biopsies that
need to be frozen for further immunohistochemical studies.
REMOVAL OF FIXATION PIGMENTS
Formalin Pigments these can be removed by immersing the sections in saturated
absolute alcohol with picric acid for 10 minutes to three hours. Then wash sections well with
water. Also, a solution of 70% alcohol containing 3 mL of ammonium hydroxide for 30
minutes to 3 hours. After treatment wash sections well in 1% acetic acid.
Mercury Pigments these can be removed by immersing the sections in Gram or Lugols
Iodine for 10 minutes, and then place the section in a 5% solution of Sodium thiosulfate for
3 minutes. Wash slides well in running water for 10 minutes.
Melanin Pigments this is removed by placing the section in a solution of Potassium
permanganate for approximately 20 minutes, followed by a solution of Oxalic acid to
remove the excess of Potassium permanganate.
Fixatives: a summary
It is clear that there is no universal fixative which will serve all requirements. Each fixative
has specific properties and disadvantages and their many different effects emphasize the
necessity for careful consideration and selection of the appropriate fixing reagent when
studies of specific cellular substances are planned. Ten per cent buffered formalin and 2.5%
Gluteraldehyde are currently the most widely used fixatives for routine light microscopy and
ultrastructural studies, but they too, have inherent disadvantages which the user should be
well conversant with. Increasing interest in tissue and cell constituents including cellular
proteins detectable by immunohistochemical techniques imposes additional requirements for
the preservation of such substances.