Definitions

 Histology:
 Science of examination of normal tissues
‫علم فحص األنسجة الطبيعية‬

 Histopathology:
 Examination of tissues for presence /absence of changes in structure due to disease
process.
Autolysis: Is a self-destructive process due to the release of autolytic enzymes
(lysosomes) from the dead cells.

Putrefaction: Occurs due to the action of bacteria that invade the tissue

Fixation: are Complex series of chemical events which differs for

different groups of substances found in tissues

Fixative: substance which prevent postmortem changes and preserves the

morphological and chemical characteristics of cells and tissues

What is the series of processes fixation?

1-dehydration 2-clearing, 3-embedding 4-cutting 5-staining

What is the Principle in the action of fixative

Fixatives act by denaturing or precipitating proteins which then form a meshwork due to
cross linking of proteins

Numeric (explain) Pre-fixation procedures & precautions:

 Dissecting table made up of hard wood with a working
surface area of 45 X 35 cm
 Wood- actual cutting surface
 Rubber pads of various dimensions- suitable
 Instrument- thin bladed knives 20-30 cm in length, scissors,
probes, scalpels, stainless-steel rule, forceps, sponge First-aid box & eye-wash
bottle
 Cutting board & instruments should be cleaned before
 Washing of the gloved hands before dissection
 Fixation of tissue- as soon as possible after death or
removal from the body
 Screw capped specimen jars containing appropriate fixatives
 Amount of fluid in the jars- 15-20 times
 Early dispatch of the specimens to histopathology laboratory

What does the Purpose of Fixation

 Preservation of cells & tissue constituents in a condition identical to
that existing during life.
 To prevent autolysis, & putrefaction
substances.
 To fortify the tissue against deleterious effects of further stages.
 To facilitate differential staining with dyes
 It should penetrate evenly and rapidly.
 It should harden the tissues
 Increase the optical differentiation of cells & tissues
 Should not cause shrinkage or swelling of the cells
 Must not react with the receptor sites & thus must not interefere with
the staining procedure
 It must be cheap and easily available.
 Good fixative is most important factors in the production of
satisfactory results in histopathology

What are the factors that help to stabilize the tissues? :

 Fresh tissue
 Proper penetration of tissue by fixatives
 Correct choice of fixatives
 No fixative will penetrate a piece of tissue
thicker than 1 cm.

Types of methods of fixation

1. Perfusion/ in vivo fixation:
2. Immersion/ in vitro fixation
3. Perfusion-immersion
4. Fixation using vapors method
recommended For dealing with specimen thicker, following
are
1. Solid organ:
 Cut slices as necessary as
but not thicker than 5 mm.

2. Hollow organ:
 Either open or fill with
fixative or pack lightly with
wool soaked in fixative.

3. Large specimen:
 It requires dissection, inject
fixative along the vessels or
bronchi as in case of lung
so that it reaches all parts
of the organs.

Methods of specimen fixation

Solid organ Hollow organ

What Properties of an ideal fixative

 Preserves tissue in their natural state and fix all tissue components.
 Make the cellular components insoluble to reagent used in tissue
processing
 Cause minimum physical and chemical alteration of the tissue and its
components
 Should penetrate a tissue quickly
 Neither shrinks nor swells
 Makes the specimen hard enough
 Raises refractive indices
 Provides full range of staining methods with great selectivity
 No rigid upper limit of fixing time
 No tendency to deteriorate
 Non-toxic, stable & safe to handle
 Non-inflammable & non-irritant
 As such no ideal fixative
 Choice depends on the cell or tissue constituent to be demonstrated
Mechanism / Action of Fixatives
 Most fixatives act by denaturing or precipitating
proteins which then form a sponge or meshwork,
tending to hold the other constituents

General classification of fixatives

1. Aldehydes (cross linking agents):
 Formaldehyde and Gluteraldehyde
 Act by creating covalent chemical bonds between
proteins of tissues- Oxidizing agents- joins with
various side chains of protein molecules & other
biomolecules- allow formation of cross linkstabilizes tissue structure.
2. Protein denaturing agents:
 Acetic acid, methyl alcohol, ethyl alcohol
 Reduce the solubility of protein without
combining with it & disrupts the
hydrophobic bonds which is needed for its
tertiary structure to form.
3. Unknown mechanism:
 Mercuric chloride (B5 fixatives)
 It increases the staining brightness & give
good nuclear detail. Good for reticuloendothelial tissue & haemopoetic tissue.
4. Picrates:
 Picric acid binds with histone & basic proteins to
form crystalline picrates with amino acid &
precipitates protein.
 5. Oxidizing agents:
 Osmium tetroxide, potassium permanganate,
potassium dichromate.

1. Physical methods of fixation

1. Heat fixation:
 The simplest form of fixation is heat. Boiling or
poaching an egg precipitates the proteins and, on
cutting, the yolk and egg white can be identified
separately.
2. 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.
3. Freeze-drying and freeze substitution:
 Is a 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

. Chemical fixation
 Chemical fixatives can be considered as members of
three major categories:
1. Coagulant fixatives:
 Coagulating such proteins maintains tissue histomorphology at the light
microscopic level.
 Dehydrant coagulant fixatives
 The most commonly used coagulating fixatives are alcohols (e.g. ethanol,
methanol) and acetone. Methanol is closer to the structure of water than
ethanol.
 Dehydrant fixatives act to remove free and bound water, causing a change to
the tertiary structure of proteins so that they precipitate, leaving the nucleic
acids relatively unchanged.
 Ultrastructure is destroyed by any of these four dehydrants due to the
extraction of lipids, and each may cause excessive shrinking of tissue
components after more than 3–4 hours of fixation.
 Each of these fixatives can be modified by adding other chemicals to produce
specific effects.
 Examples of dehydrant coagulant fixatives
1. Absolute Ethanol
2. Absolute Methanol
3. Ethanol, 95%
4. Acetone
 Methanol is useful for touch preparations and smears,
especially blood smears.
 Other types of coagulant fixative
 Acidic coagulants such as picric acid and trichloroacetic
acid change the charges on the ionizable side chains
 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.

2. Non-coagulant cross-linking fixatives

 Several chemicals were selected as fixatives
secondary to their potential actions of forming
cross-links within and between proteins and
nucleic acids as well as between nucleic acids and
proteins.
 Examples of chemical fixatives include:
 Formaldehyde, glutaraldehyde, and other
aldehydes, e.g. chloral hydrate and glyoxal, metal
salts such as mercuric and zinc chloride, and other
metallic compounds such as osmium tetroxide.
1. Simple Fixatives
 1. Formaldehyde fixation:
 Formaldehyde in its 10% neutral buffered form (NBF) is the most common
fixative used
in diagnostic pathology.
 Pure formaldehyde is a vapor that, when completely dissolved in water, forms
a solution
containing 37–40% formaldehyde; this aqueous solution is known as ‘100 %
formalin’.
 The time to saturation of human tissues with active groups by formalin is
about 24 hours,
but cross-linking may continue for many weeks.
 When formaldehyde dissolves in an un-buffered aqueous solution, it forms an
acid
solution (pH 5.0 - 5.5) because 5 - 10% of commercially available formaldehyde
is formic
acid.
 Stabilizer – 10 - 14% methanol
 On storage becomes acidic by formation of formic acid.
 Turbid on keeping - paraformaldehyde.
 Yellow - contaminated with ferric iron from metal containers (Positive
Prussian blue
reaction).
 Fixes protein, lipids well preserved.
 Favors staining of acidic structures like nuclei with basic dyes
 Diminishes effect of acid dyes on basic structures like cytoplasm.
Advantages of Formaldehyde
 Formaldehyde primarily preserves peptide protein bonds
and the general structure of cellular organelles.
 Easy to prepare
 Relatively stable
 It allows subsequent use of most staining procedures.
 Frozen sections can be prepared with ease.
 Fixes lipids for frozen sections and tissue enzymes.
 Penetrates tissues well.
 Beneficial hardening with little shrinkage
 Natural tissue colors are retained.
 Does not require washing before processing.
 Best fixative for nervous system.

 Disadvantage of Formaldehyde
 Using acid formalin for fixation is the formation of a
brown-black pigment with degraded hemoglobulin.
 Unpleasant vapour irritant to the nose, the eyes and
mucous membranes
 Irritation to respiratory epithelium
 Formalin dermatitis
 Formation of precipitate of paraformaldehyde which
can be prevented by adding 11- 16 % methanol.
 It causes shrinkage of collagen.
 It suspected to contain cancer producing agents.
 Formalin pigment
 Brown, granular material, extracellular, birefringent
 Progressive in deposition
 Often seen after several days
 Action of acid formalin on blood
 Avoided by using buffered formalin.
 Removed by treatment with saturated alcoholic solution of
picric acid for 20mins
 For routine histopathology, 10% neutral buffered formalin
(NBF) is frequently used for initial fixation and for the first
station on tissue processors.

2. Glutaraldehyde fixation
 Glutaraldehyde is a bifunctional aldehyde that
probably combines with the same reactive groups
as does formaldehyde.
 In aqueous solutions glutaraldehyde polymerizes,
forming cyclic and oligomeric compounds.
 It is oxidized to glutaric acid.
 To aid in stability, it requires storage at 4°C and at
a pH of around 5.
 Used for Electron Microscopy with osmium
tetroxide.

 Advantage Glutaraldehyde fixation:

 Most efficient cross linking agent for collagen
 More rapid fixation than formalin.
 Disadvantage:
 False positivity with Periodic Acid Schiff’s (PAS).

3. Osmium tetroxide fixation

 Pale yellow.
 Toxic solid
 Soluble in water as well as non-polar solvents and can
react with hydrophilic and hydrophobic sites
including the side chains of proteins, potentially
causing crosslinking.
 Excellent preservation of detail of single cells
 Use as a secondary fixative for electron microscope
examinations
 Used to stain lipids (myelin) in frozen sections.
 Storage in dark, cool place.

 Disadvantages Osmium tetroxide fixation:

 Uneven penetration for pieces more than 2-3mm
 Vapor is irritating, causes conjunctivitis
 Causes tissue swelling which is reversed during
dehydration steps.
 Swelling can be minimized by adding calcium or
sodium chloride to osmium-containing fixatives.
4. Mercuric chloride
 White crystalline substance.
 Powerful protein precipitant, fixes both nucleus &
cytoplasm well favoring its staining.
 Enhancing the staining properties of tissues,
particularly for trichrome stains.
 Conjunction with other fixatives.
 Hardens tissue
 Radio-opaque.

 Disadvantages of Mercuric chloride:

 These fixatives penetrate slowly, so specimens must be thin
 Mercury and acid formaldehyde hematein pigments may deposit in tissue after
fixation.
 Formation of deposits of intensely black precipitates of mercuric pigment in
the tissues.
 Should be dissolved in distilled water to prevent the precipitation of mercury
salts.
 These precipitates can be readily removed by a Lugol’s iodine step in the
staining procedure, followed by bleaching of the section in sodium
hypochlorite solution (Hypo).
 Mercury-based fixatives are toxic and should be handled with care.
 Corrosive to metals, they should not be allowed to come into contact with
metal.
 Pollution to environment, mercury-containing chemicals are an environmental
disposal problem.

2. Compound fixatives
 Other agents may be added to formaldehyde to produce specific effects that
are
not possible with formaldehyde alone.
 The dehydrant ethanol, for example, can be added to formaldehyde to produce
alcoholic formalin.
 This combination preserves molecules such as glycogen and results in less
shrinkage and hardening than pure dehydrants.
 Compound fixatives are useful for specific tissues, e.g. alcoholic formalin for
fixation of some fatty tissues, such as breast to aid in identifying lymph nodes
embedded in fat.
 Some combined fixatives including alcoholic formalin are good at preserving
antigen immunorecognition.
 Types of Mercuric chloride containing fixatives
1. Zenker's Solution:
 M.C. Fixatives rapidly penetrates tissues and
permit excellent staining of nuclei and connective
tissues.
 Disadvantage Mercuric chloride containing fixatives :
 Causes hardening of tissues
 Fixed tissue should be washed overnight in
running tap water before processing.

2. Helly’s solution:
 By adding formalin to Zenker’s solutions the beneficial
effects of both fixatives are combined, minimizing their
disadvantages.
 Staining of nucleus, cytoplasm and connective tissue is
good with Helly’s fluid.
 Fixation time 12-24 hours.
3. Schaudinn’s solution
4. Ohlmacher’s solution
5. Carnoy-Lebrun solution
6. B5 fixative
.
Dichromate-containing fixatives:
 Fixatives containing chromate at a pH of 3.5–5.0 make proteins
insoluble without coagulation.
 More acidic pH – fixes nucleus & cytoplasm
 Chromate is make unsaturated but not saturated lipids insoluble upon
prolonged (>48 hours) fixation and hence mitochondria are well
preserved by dichromate fixatives.
 Dichromate-containing fixatives have primarily been used to prepare
neuroendocrine tissues for staining, especially normal adrenal medulla
and related tumors (e.g. phaeochromocytomas).
 Wash in running water after to prevent formation of insoluble
precipitate.
 Prolonged exposure causes tissue to become brittle.
 Use as mordant.

 Types of dichromate-containing fixatives:

1. Chromic acid
2. Miller’s or Möller’s solution
3. Möller’s or Regaud’s solution
4. Orth’s solution.
 Picric acid -containing fixatives
 Many picric acid fixatives require a saturated
aqueous solution of picric acid.
 Aqueous picric acid 2.1% will produce a
saturated solution and 5% picric acid a saturated
solution in absolute ethanol.
1. Bouin’s fluid
2. Hollande’s solution
3. Fixatives for DNA, RNA, and protein analysis.

. Histochemical fixatives
 These are used to demonstrate the chemical constituents
of the cell.
 Preserve the constituent to be demonstrated & its
morphological relationships.
 Without affecting the reactive groups & reagent to be
used in its visualization.
 Best – cryostat cut sections of rapidly frozen tissue.
 Cold acetone – where enzymes are to be studied especially
phosphatases
 Vapour fixatives like formaldehyde, acetaldehyde,
gluteraldehyde, acrolein to fix cryostat cut sections of
fresh tissue & blocks of frozen dried tissue.
The most commonly fixatives used in histopathology lab
1. 10% Formalin
2. Carson’s modified Millonig’s phosphate:
 Is buffered with sodium monobasic phosphate and sodium
hydroxide.
3. 10% Formal calcium:
 Recommended for the preservation of lipids especially
phospholipids.
4. 10% Formal saline
5. Formalin, buffered saline
6. 10% Zinc formalin (unbuffered):
 It gives improved results with Immunohistochemistry
(IHC).

Factors affecting the quality of fixation

1. Buffers & hydrogen ion concentration (pH)
2. Duration of fixation and size of specimens
3. Temperature
4. Concentration
5. Osmolality of fixatives and ionic composition
6. Additives
7. Volume ratio
8. Choice of fixatives
9. Penetration
10. Tissue storage

1. Buffers & hydrogen ion concentration (pH)

 The most reliable way of achieving optimal formalin
fixation is through its buffering at pH 7.2–7.4 (i.e. neutral
buffered formalin).
 The choice of specific buffer depends on the type of
fixative and analyte.
Commonly used buffers are:
 Phosphate, Cacodylate, Bicarbonate, Tris, and Acetate.
 It is necessary to use low salt-buffered formalin in the
new complex tissue processors in order to keep the
machine ‘clean’, and reduce problems in its operation.

2. Duration of fixation and size of specimens

 Blocks should be small or thin (3 – 5 mm) to ensure adequate penetration.
 The depth (d) reached by a fixative is directly proportional to the square root
of
duration of fixation (t) and expressed this relation as:
d = k √t
 The constant (k) is the coefficient of diffusability, which is specific to each
fixative.
 NBF, will penetrate tissue to the depth of approximately 1 mm in one hour
 Gross specimens should not rest on the bottom of a container of fixative: they
should be separated from the bottom by wadded fixative-soaked paper or cloth,
so allowing penetration of fixative or processing fluids from all directions.
 When surgical specimens are to be processed to paraffin blocks, the time of
penetration by fixative is more critical.
 Proteins inactivate fixatives, especially those in blood or bloody fluids.
 Bloody gross specimens should therefore be washed with saline prior to being
put into fixative.
 The fixative volume should be at least 15 times the volume of the tissue
specimen for optimal, rapid fixation i.e 1:15.

3. Temperature of fixation
 Most tissues fixed at room temperature.
 The diffusion of molecules increases with rising
temperature due to their more rapid movement and
vibration; i.e. the rate of penetration of a tissue by
formaldehyde is faster at higher temperatures.
 Microwaves therefore have been used to speed
formaldehyde fixation by both increasing the temperature
and molecular movements.
 Increased vapor levels are a safety problem.
 Most chemical reactions occur more rapidly at higher
temperatures and therefore formaldehyde reacts more
rapidly with proteins.
 Electron microscopy & histochemistry at 0 - 4˚C.

4. Concentration of fixative
 Different concentrations have different effects on
morphology.
 Effectiveness and solubility primarily determine the
appropriate concentration of fixatives.
 Concentrations of formalin above 10% tend to cause
increased hardening and shrinkage.
 Higher concentrations result in formalin being present in
its polymeric form, which can be deposited as white
precipitate.
 Ethanol concentrations below 70% do not remove free
water from tissues efficiently.
 Effects subsequent staining.
5. Osmolality
 6. Osmolality of fixatives and ionic composition
 The osmolality of the buffer and fixative is important.
 Hypertonic solutions lead to shrinkage.
 Hypotonic solutions cause swelling of cells & poor fixation.
 The best morphological results are obtained with solutions that are
slightly hypertonic (400–450 mOsm)
 The osmolality for 10% NBF is about 1500 mOsm.
 The ionic composition of fluids should be as isotonic as possible to the
tissues.
6. Additives
 The addition of electrolytes and non-electrolytes to fixatives improves
the morphology of the fixed tissue.
 These additives include calcium chloride, potassium thiocyanate,
ammonium sulfate, and potassium dihydrogen phosphate.
 The electrolytes may react either directly with proteins causing
denaturation, or independently with the fixatives and cellular
constituents.
 Fixatives buffered with electrolytes such as phosphates may cause
problems with some processors due to precipitation of the salts.
 The addition of non-electrolyte substances such as sucrose, dextran, and
detergent improve fixation.

Secondary fixation
 Tissues fixed with two fixatives in
succession.
 Improved preservation & staining.
 Tissues fixed in buffered formalin –
fixed with mercuric chloride.
 Tissues fixed with gluteraldehyde is
post fixed with osmium tetroxide for
electron microscopy.
Post chromatization
 Treatment of tissues with 3% potassium
dichromate following normal fixation.
 Before processing – tissue in dichromate
solution for 6- 8days
 After processing – for 12- 24 hrs
 Both followed by washing in running water
 Employed to mordant tissues.
 Mitochondria & myelin demonstrated.
 Improved preservation and staining of
elements.