DEPARTMENT OF PATHOLOGY

GANDHI MEDICAL COLLEGE, BHOPAL

SEMINAR
“FIXATIVES IN HISTOPATHOLOGY”

DATE PRESENTED BY
29-01-2018 DR. ABHINAV JUNWAL
CONTENTS :

1. Introduction
2. Methods of Fixation
3. Physical Methods
4. Coagulant Fixatives
5. Non Coagulant Fixatives
6. Factors Affecting Fixation
7. Fixation for selected Individual Tissues
8. Special Formulae fixatives
 INTRODUCTION
Preparation of histological specimen for examination undergo
the following steps :
1. Fixation
2. Tissue Processing :
 Dehydration .
 Clearing .
 Impregnation .
 Embedding .
3. Microtoming .
4. Cutting .
5. Staining .
 TISSUE FIXATION
Once tissues are removed from the body, they undergo a process of self-destruction or
autolysis which is initiated soon after cell death by the action of intracellular enzymes
causing 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 fibre and collagen.

Fixation is a complex series of chemical events which preserves the tissues in as close a
life like state as possible by preventing their autolysis and putrefaction. During this
process, the semi fluid state of the cell is converted into a semisolid state thus
maintaining, the morphology and structural details of the tissue.

The major objective of fixation in pathology is to maintain

clear and consistent morphological features.
 PRINCIPLE OF FIXATION

The fixative brings about crosslinking of

proteins which produces denaturation or
coagulation of proteins so that the semifluid
state is converted into semisolid state; so that it
maintains everything in vivo in relation to each
other. Thus semisolid state facilitate easy
manipulation of tissue.
 OBJECTIVES OF AN IDEAL FIXATIVE
1. Preserve tissue in a life-like state.

2. Prepare tissues for subsequent processing

3. Prevent osmotic damage

4. Prevent shrinkage and swelling

5. Prevent any change in volume or shape during the subsequent procedures

6. Preserve all cell constituents

7. Harden the tissues allowing easy sectioning

8. Convert the semi fluid consistency of cells to an irreversible semi solid consistency (sol
to gel)

9. Render tissue components resistant to extraction by water and organic solvents

10. Optimum Optical differentiation.

 TYPES OF FIXATION
1. Immersion fixation
2. Perfusion fixation
3. Vapour fixation
4. Coating/Spray fixation
5. Freeze drying
6. Microwave fixation/Stabilization
 METHODS OF FIXATION
PHYSICAL METHODS
 Heat Fixation :
• Simplest form of fixation.
• In histopathology heat is primarily used to accelerate other forms of fixation as well as the
steps of tissue processing.

 Microwave Fixation :
• Speeds fixation of some gross specimens and histological sections from more than 12 hours to
less than 20 minutes.
• Produce harmful vapours so requires a microwave processing system.

 Freeze-drying and freeze substitution :

• Tissues are cut into thin sections, immersed in liquid nitrogen, and the water is removed in a
vacuum chamber at −40°C.
• 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.
• Used primarily in the research environment
 CHEMICAL METHODS
DEHYDRANT COAGULANT FIXATIVES :

Ethanol and Methanol -

• Alcohol acts as reducing agents, become oxidized to acetaldehyde and then to acetic
acid.
• They are slow to penetrate, hardens and shrinks the tissue.
• Penetrates rapidly in presence of other fixative hence in combination e.g. Carnoy's
fixative is used to increase the speed of tissue processing.
• Ethanol preserves some proteins in relatively undenatured state so that it can be used for
immunofluorescence or some histochemical methods to detect certain enzymes. It is a
fat solvent hence it dissolve fats and lipids
• Methyl alcohol is used for fixing blood and bone marrow smears.

Acetone –
• Cold acetone is sometimes used as a fixative for the histochemical demonstration of
some tissue enzymes like phosphatases and lipases.
• Its mode of action as fixative is similar to that of alcohol
 OTHER COAGULANT FIXATIVES
PICRIC ACID
• It produces marked cells shrinkage hence it is not used alone.
• It has to be stored in a damp place because of its explosive nature it is preferably stored
under a layer of water.
• It penetrates well and fixes rapidly.
• It precipitates proteins and combines with them to form picrates some of the picrates are
water-soluble so must be treated with alcohol before further processing where the tissue
comes into contact with water.
• All the tissues fixed in picric acid containing fixatives should be thoroughly washed to
remove the yellow discolouration to ensure proper staining of tissue sections.
• If the fixative is not removed by washing thoroughly with time even the embedded tissue
looses its staining quality.

ACETIC ACID
It causes the cells to swell hence can never be used
alone but should be used with fixatives causing cell shrinkage.
 NON COAGULANT CROSS LINKING FIXATIVES

Cross linking fixatives act by creating covalent chemical bonds between proteins in
tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to
the tissue. Examples 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.

 FORMALEHYDE 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 ‘formalin’. The usual ‘10% formalin’
used in fixation of tissues is a 10% solution of formalin; i.e., it contains
about 4% weight to volume of formaldehyde.
• It preserves the proteins by denaturation and forming crosslinkage
with them and the tissue component.
• The commercial formalin becomes cloudy on standing especially
when stored in a cool place due to formation of precipitate of
paraformaldehyde which can be filtered.
• Formalin on prolonged exposure can cause either dermatitis its
vapour may damage the nasal mucosa and cause sinusitis.

• Time required for fixation.

At room temperature - 12 hours

For small biopsies - 4-6 hours
At 65°C fixation occurs in - 2 hours

 GLUTARALDEHYDE FIXATION:
• causing deformation of the alpha-helix structures in proteins.
• larger molecule, and may not penetrate thicker tissue specimens as effectively as
formaldehyde
• Thus small blocks of tissue are required.
• may offer a more rigid or tightly linked fixed product—its greater length and two
aldehyde groups allow it to 'bridge' and link more distant pairs of protein molecules.
• It causes rapid and irreversible changes, fixes quickly, is good for electron microscopy,
fixes well at 4oC, and gives best overall cytoplasmic and nuclear detail.

• However it is not ideal for immunohistochemistry staining

• Some fixation protocols call for a combination of formaldehyde and glutaraldehyde, so

that their respective strengths complement one another.

 OSMIUM TETROXIDE FIXATION :

• It is a strong oxidizing agent and brings about fixation by forming cross links with proteins.
• It gives excellent preservation of details of a cell, therefore exclusively used for electron
microscopy.
• It fixes fat e.g. myelin and also demonstrates fat when 0.5-2% aqueous solution is used it
gives a black colour to fat.
 MERCURIC CHLORIDE FIXATION :

 Its a very good salt employed in fixing but is rarely used alone because it causes
shrinkage of the tissue.
 It brings about precipitation of the proteins which are required to be removed before
staining by using potassium iodide in which they are soluble.
 if the tissue is more than 4 mm, then it hardens the tissue at the periphery whereas the
centre remains soft & under fixed.
 It penetrates rapidly without destroying lipids.
 It neither fixes nor destroys carbohydrates. Treatment of the tissue with mercuric
chloride brings out more brilliant staining with most of the dyes.
 Tissues fixed with mercuric chloride containing fixatives contain black precipitates of
mercury which are removed by treating with 0.5% iodide solution in 70% ethanol for 5-
10 minutes, sections are rinsed in water, decolourized for 5 minutes in 5% sodium
thiosulphate and washed in running water.
DICHROMATE AND CHROMIC ACID FIXATION :
 Pottasium dichromate has a binding effect on protein similar to that
of formalin. Following fixation with Potassium dichromate tissue must
be well washed in running water before dehydration
 Chromic acid precipitates all proteins and preserves
carbohydrates. Tissues fixed in chromic acid also require thorough
washing with water before dehydration.
 Dichromate-containing fixatives have primarily been used to
prepare neuroendocrine tissues for staining, especially normal
adrenal medulla and related tumors (e.g. phaeochromocytomas).
However, reliance on the chromaffin reaction used to identify
chromaffin granules following dichromate fixation has greatly
diminished, being replaced by immunohistochemistry to a range of
neuroendocrine markers.
Factors affecting Fixation:
 1. Temperature – Standardized fixation is carried out at room
temperature. However, for electron microscopy and some
histochemical procedure, fixation is usually at 0 – 4 degrees Celsius.
Lower temperatures autolysis is slowed down allowing a more life-like
appearance of tissues.

 2. Size of specimen – Larger tissue size will be fixed slowly. Thus, large
specimens are opened and washed of contaminant or sliced thinly. .
The fixative should be at least 20 times the volume of the specimen.
Cut sections should be 1-4 mm Thickness

 3. Change in volume – The volume changes in tissue with fixation, as

the tissue shrinks by 33%. This is evident as the tissue show larger nuclei
and cells in frozen sections (unfixed).
4. pH and Buffers – The hydrogen ion concentration varies between
fixatives, Should be kept in between pH 4-9.
The buffer systems maintain this physiological range of pH level
(e.g.; phosphate, acetate, bicarbonate). The buffer should have
the following properties;
• Do not interfere with fixative
• Do not inhibit enzymes

5. Osmolality – The best result is obtained by using slightly hypertonic

solutions (isotonic solutions adjusted by using sucrose). Hypertonic
solutions give rise to cell shrinkage. Hypotonic solutions result in cell
swelling and poor fixation.

6. Concentration of fixative

7. Duration of fixation - As a general rule 1hour per1mm

 FIXATION FOR SELECTED INDIVIDUAL TISSUES
 EYES - Fixed in NBF, usually for about 48 hours.
 BRAIN - This fixation takes at least 2 weeks. Adickes et
al. (1997) proposed a perfusion technique which allows
all of the above to be accomplished and the report
issued in 5–6 days.
 BREAST -
 Fixed in 10% NBF for between a minimum of 6–8 hours and a
maximum of 72 hours, and should be sliced at 5mm intervals.
 Time from tissue acquisition to fixation should be as short as
possible in order to prevent lysis of clinically important
biomarkers, such as estrogen receptors, progesterone
receptors and the human epidermal growth factor receptor-2
(HER2).
 LUNGS - Lung biopsies are typically fixed in NBF.
 LYMPHOID TISSUE –
• Special care should be taken with all lymphoid tissue, as many
organisms (e.g. Mycobacterium tuberculosis and viruses) may
sequester themselves in the lymphoid reticular system.
• Lymph node is fixed in NBF, though some laboratories fix part of the
tissue in B5 or zinc.
 TESTIS - Biopsies of the testes are fixed routinely in NBF.
 RENAL BIOPSIES –
• NBF for routine histology.
• Buffered glutaraldehyde (pH 7.3) for ultrastructural analysis.
• Snap frozen in isopentane and liquid nitrogen for
immunofluorescence examination.
 IMPORTANT FORMULATION FIXATIVES
 Formalin based fixatives –
1. Neutral buffered 10% formalin:
 Tap water
 900 ml Formalin (40% 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

2. Carson’s modified Millonig’s phosphate buffered formalin:

 Formaldehyde (37–40%)
 10 ml Tap water
 90 ml Sodium phosphate
 Monobasic 1.86 g
 Sodium hydroxide 0.42 g
3. Formal (10% formalin) calcium acetate :
 Tap water 900 ml
 Formaldehyde (40%) 100 ml
 Calcium acetate 20 g
This is a good fixative for preservation of lipids.

4. Formal (10% formalin) saline :

 Tap water 900 ml
 Formaldehyde (40%) 100 ml
 Sodium chloride 9 g

5. Formal (10% formalin) zinc – unbuffered :

 Tap water 900 ml
 Formaldehyde (40%) 100 ml
 Sodium chloride 4.5 g
 Zinc chloride or (zinc sulfate) 1.6 g (or 3.6 g)
Zinc formalin is reported to be an excellent fixative for immunohistochemistry.
 Mercuric Fixatives –
1. Zenker’s solution - This is a good fixative for bloody (congested) specimens.
2. Helly’s solution - excellent for bone marrow extramedullary hematopoiesis and
intercalated discs.
3. Schaudinn’s solution
4. Ohlmacher’s solution
5. Carnoy-Lebrun solution
6. B5 fixative - Frequently used for bone marrow, lymph nodes, spleen, and other
hematopoietic tissues.

 Dichromate Fixatives –
1. Miller’s or Möller’s solution
2. Möller’s or Regaud’s solution
3. Orth’s solution
 Picric Acid Fixatives –
1. Bouin’s solution - an excellent general fixative for connective tissue stains.
2. Hollande’s solution - A useful fixative for gastrointestinal biopsies and endocrine tissue.

 For Metabolic Bone Disease -

 Phosphate buffer
o Tap water 1000 ml
o NaH2PO4·H2O 1.104 g
o NaHPO4 (anhydrous) 4.675 g
 Fixative
o Phosphate buffer 900 ml
o Formaldehyde (40%) 100 ml
Adjust pH to 7.35.
 FIXATION FOR FATTY TISSUE

 Bouin’s solution 75 ml
 95% ethanol 25 ml
May require up to 48 hours for good sections of
lipomas or well-differentiated liposarcomas.
THANK YOU