FIXATION OF

HISTOLOGY SAMPLES
DR. DAMPTEY OBED KYEM (BSC. MBCHB)
SCOPE
INTRODUCTION
• Fixation is the process by which the cells in the tissue are fixed in a
chemical and physical state, and all the biochemical and proteolytic
activities within the cells are prevented so that the cells or tissues can
resist any morphological change or distortion or decomposition after
subsequent treatment with various reagents.
• The fixation helps to maintain the tissue nearest to its original state in
the living system
Aims of fixation
• To preserve the tissue nearest to its living state
• To prevent any change in shape and size of the tissue at the time of
processing
• To prevent any autolysis
• To make the tissue firm to hard
• To prevent any bacterial growth in the tissue
• To make it possible to have a clear stain
• To have a better optical quality of the cells
Ideal fixative
An ideal fixative should have the ff qualities
• Prevention of autolysis of the cells or tissue
• Prevention of decomposition of the tissue by bacteria
• Maintaining the volume and shape of the cell as far as possible
• Consistently high-quality staining, mainly routine stains such as
Hematoxylin and eosin stain and Papanicolaou`s stain
• Rapid action
• Cheap
• Non-toxic
Tissue changes in fixation
• Volume changes: fixatives may change the volume of cells. Some fixatives such
as Osmium tetroxide causes cell swelling. the volume change may be due to
i. Altered membrane permeability
ii. Inhibition of the enzymes responsible for respiration,
iii. Change of transport of Na+ ions
Formaldehyde may cause shrinkage of volume by 33%
• Hardening of tissue: the fixation changes the consistency of the tissue, and
some amount of hardening occurs due to fixation
• Interference of staining: fixation may cause hindrance to the staining of
enzymes. Formadehyde inactivates 80% of ribonuclease enzyme. Osmium
tetroxide is known to inhibit hematoxylin and eosin staining
• Changes in optical density by fixation: the fixation may cause the
change in optical density of the nuclei, and the nuclei may look
condensed and hyperchromatic
Types of fixation
• The fixatives can be classified based on the ff criteria
• Nature of fixation
• Chemical properties
• Component present
• Action on tissue protein
ypes of fixative Classification
Nature of fixation Immersion fixation
Coating fixation
Vapour fixation
Perfusion fixation
Freeze-drying
Microwave fixation

Chemical properties Aldehyde: formaldehyde, gluteraldehyde

Oxidising agent: Osmium tetroxide
Protein denaturing agent: Ethyl alcohol, methyl
alcohol
Cross-linking agents: Carobiimide
Miscellaneous: Picric acid

Component present 1. Simple (only one chemical present

i. Formaldehyed
ii. Ethyl alcohol
iii. Glutaradehyde
iv. Picric acid
v. Osmium tetroxide
2. Compound (more then one chemical present)
i. Bouins fluid
ii. Carnoy`s solution

Action on protein Coagulative: ethyl alcohol, picric acid

Essential precautions for fixation
• For proper fixation;
1. The tissue should be free from excessive blood before putting it into a
fixative
2. Tissue should be thinly cut in 3-5mm thickness
3. The amount of fixative fluid should be 20 times more than the tissue volume
4. The tissue with fixative should be in a tightly screw-capped bottle
5. Always check the colour of formalin. It should be clear, and if there is any
change of colour, then the solution should be filtered or mildly heated
6. Never heat the fixative solution with the intention of rapid fixation as it may
cause tissue shrinkage
Mechanism of fixation
A. Dehydraton and coagulation of protein
• methanol and ethanol are commonly used coagulative fixatives.
• They remove water from the tissue and cause destabilizing of the hydrogen
bonds and disrupting the tertiary structure of the protein.
• However, the secondary structure of the protein is maintained
• Ethanol is a relatively stronger dehydrating agent than methanol
• The ethanol and methanol start work froom 60% to 80% coc. respectively.
• Disadvantages
• Shrinkage of the cells
• Removal of the soluble substances from the tissue
Mech. Of fixation ctd
B. cross-linking fixatives
1. Formaldehyde: formaldehyde in an aqueous solution combines with water to form
methylene hydrate, a methylene glycol:
CH2O+H2O=OHCH2OH
Formaldehyde+ water=methylene glycol
This methylene glycol may react with water molecules and form a polymer known as polyoxy-
methylene glycol in a longstanding position.
This again depolymerized in methylene glycol in a neutral buffer system. Formaldehyde reacts
with various protein side chains and forms hydroxyl methyl side group
These compounds are highly reactive, and subsequently, cross-linking occurs by creating a
methylene bridge. This initial reaction of the hydroxymethyl side chain is the primary reaction,
and the subsequent intermolecular and intramolecular cross linking of the molecules occurs as
a slow-growing process. This ultimately prodecs an insoluble product
• 2. Glutaraldehyde: it has to aldehyde groups separated by three
methylene bridges. The aldehyde group reacts with an amino group
of the protein, predominantly lysine.
• When one aldehyde group reacts with the amino group, the other
free aldehyde group may help with cross-linking.
• Glutaraldehyde rapidly and irreversibly corss-links the protein
• The penetration of glutaraldehyde is slower than formaldehyde
3. Osmium tetroxide (OsO4): is mainly used as a fixative in electron microscopy.
• Alone or in combination
• Causes the oxidation of unsaturated bonds in the biological tissue, particularly
lipid.
• Converts the unsaturated fatty acid into a stable product= glycol osmate
• The tetravalent Os becomes hexavalent in this rxn
• Osmic monoester formed in this rxn is readily hydrolised to diol and osmic
acid.
• Osmium tetraoxide may react with unsaturatedcarbon atoms of the lipids and
may cross-link
Factors affecting fixation
1. Hydrogen ion concentration (pH): most fixatives work better at neutral pH (6-
8). At high pH, the NH2 group of amino acid is converted to NH3, and the rxn
btn aldehyde groups of the fixative is reduced. Commonly used buffers to
maintain ph are phosphate, bicarbonate, Tris and acetate
2. Temperature: room temp is alright for tissue fixation and there is no
difference in cell morphology from0-45*C. at higher temp, the vibration and
movement of the molecules are increased. This increases the penetration rate
of the fixative within the tissue and accelerates the fixation process
3. duration of fixation: the depth of penetration of fixative is directly
proportional to the square root of the time of fixation. The diffusibility of diff
fixatives may also vary. D=k where D=depth of penetration; T=time duration
k=coefficient of diffusion of the fixative
4. Osmolarity of the fixative solution: hypertonic fixative solution causes
shrinkage of the cell, whereas the hypotonic fixative solution induces swelling
of the cells. Electrolytes (0.9% NaCl) or sucrose may be added to the fixative to
maintain the osmolarity of the fixative to maintain the osmolarity. Preferred
osmolarity=400-450milliosmole
5. Concentration: very low fixative concentration prolongs the fixative time,
and higher concentration causes rapid fixation. However the higher conc of
fixative may cause tissue hardening, tissue shrinkage and artefactual changes.
Optimal conc of formaldehyde is 10%.
6. Agitation: agitation increases penetration rate and, therefore, rapidity of
fixation. Optimum agitation is needed as slow agitation may not affect fixation,
whereas rapid agitation may have detrimental effect on delicate tissue
Commonly used fixatives
• READ ON
• Formaldehyde
• Glutaraldehyde
• Osmium tetroxide
• Methyl and Ethyl alcohol
• Acetone
• Bouin`s fixative
• Mercury salt-containing fixatives (Zenker`s fluid, Helly`s fluid, B5
fixatives)
FIXATIVES OF CHOICE
Choice of fixative based on technique
Technique Fixative of choice

Routine histopathology 10% neutral buffered formalin

Electron microscopy Glutaraldehyde solution or osmium tetroxide

Immunohistochemistry 10% neutral buffered formalin, Alcohol formalin

Immunofluorescence Unfixed cryostat

Enzyme histochemistry Fresh frozen section

Fixative of choice according to tissue
Tissue Fixative Time
Day to day sample (routine) 10% buffered formalin Small tissue: 6hr
Large tissue: 12-24hrs
Lymph node B5 solution 18hrs
Gastrointestinal tract 10% buffered formalin 6hrs
Testis 10% buffered formalin 6hrs
Or
Bouin`s fluid
Bone marrow Bouin`s fluid 3hrs
Spleen Zenker 6hrs
Eye 10% buffered formalin 48hrs
Brain Formol saline Week
Kidney biopsy Neutral buffered formalin 6hrs
Uterus Neutral buffered formalin 24hrs
Fixative of choice for different substances
Target substance Fixative of choice
Protein 10% buffered formalin
Lipid Frozen section or Osmium tetroxide
Glycogen Alcohol based fixative
Mucopolysaccharide Frozen section
Enzyme Frozen section
DNA and RNA Alcohol based fixative
Iron Alcohol based fixative
FIXATIVE ARTIFACT
A. FORMALIN PIGMENT: unbuffered formalin is kept for long time is converted
into formic acid that reacts with haemoglobin derivatives of the blood and
produces acid formaldehyde hematin which is insoluble brownish black granular
refractile birefringent pigment
Removing the pigment
1. Picric acid method
i. The sections is immersed in xylene followed by alcohol to bring in water
ii. Subsequently the section is immersed in 1.8% picric acid in absolute ethyl
alcohol for 15min
iii. It is then washed thoroughly
iv. Section is re-stained
Artifacts ct
2. Schridde`s Method
i. The section is immersed in xylene followed by alcohol to bring in water
ii. Keep the section for 30mins in 100ml of 75% alcohol and 0.5ml of 25%
liquor ammonia
iii. Wash thoroughly
3. verocay`s Method
iv. The sections is immersed in xylene followed by alcohol to bring in water
v. Keep the section for 10mins in 100ml of 80% alcohol and 1ml of aqueous
potassium hydroxide
vi. Wash thoroughly
Artifacts ct.
B. Mercury Pigments
When tissue is fixed by mercuric chloride, it produces a dark brown irregular
deposit
Location- throughout the tissue
Removal- the application of iodine converts it into mercuric iodide which is
removed by sodium thiosulphate
C. Fuzy staining
Here the nuclear and cytoplasmic details are onscured and the section looks
fuzzy or hazy
Cause- improper fixation either due to insufficient fixative or too little time in
fixative
Artefacts ct.
D. Prolonged fixation
This cause shrinkage of the tissue followed by separation. The tissue
may show holes or empty spaces within it
E. Dichromate Deposit
If the tissue is not properly washed after dichromate fixation, then the
chromium salt may form. This chrome salt reacts with alcohol and
insoluble yellow-brown precipitate may appear
Removal- by 1% hydrochloric acid in 70% alcohol for 30mins
Medaase