HISTOPATHOLOGY LEC

LECTURE 6: FIXATION
KYLE VINCI P. SOLANO, RMT, MD
JUNE 21, 2021
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FIXATION ○ Meaning, there are a lot of dissolved substances

● Fixes a piece of tissue in a certain point of time in which within the solution in a cellular environment
individual cells of the tissue will not further do their cellular ○ Hypoosmolar = swell
processes ○ Formalin is most of the time hyperosmolar so it
○ Ex. A cell that is secreting mucin. Once a fixative tends to shrink
is introduced, it will no longer produce mucin. ● Hardening of tissue
○ They make the proteins individual proteins within ○ Mild degree hardening may occur.
the cells. ○ There are tiny gaps within the cell scaffolding of
○ They make it so that it becomes rigid, forcing it to the cell trying to compress it to make a more solid
have bonds with the adjacent proteins. structure
○ It makes the structure more stable and then there ● Interference of staining
will be no longer cellular process. ○ Inhibits routine stain: Osmium tetroxide inhibits
○ Meaning, ATP won’t be used up and there will be haematoxylin and eosin staining.
no apoptosis. ○ Osmium tetroxide is very rare and expensive
● Fixative kills the cells but it fixates it in histologically ficative and is only used for electron microscopy
wherein we can still see its structure ● Changes of optical density by fixation
● Kills bacterial decomposition also known as putrefaction ○ Nuclei may look hyperchromatic.
● First step in histopathologic process
● The first step is always the hardest because there are a lot
of things that can go wrong. Types of fixative Classification
○ Ex. Specimen was not immediately fixed
■ The metabolic processes within the ● Immersion fixation
tissue will still continue and will undergo ● Coating fixation
apoptosis. A. Nature of ● Vapour fixation
■ Thus, we can no longer appreciate the fixation ● Perfusion fixation
histologic picture of the specimen ● Freeze-drying
A. Aims of Fixation ● Microwave fixation
● To preserve the tissue nearest to its living state
● To prevent any change in shape and size of the tissue at ● Aldehyde: formaldehyde,
the time of processing glutaraldehyde
● Oxidising agent: osmium
● To prevent any autolysis
tetroxide
○ Happens when u use up all the ATP B. Chemical
● Protein denaturing agent:
● To make the tissue firm to hard properties ethyl alcohol, methyl alcohol
● To prevent any bacterial growth in the tissue ● Cross-linking agents:
● To make it possible to have clear stain carbodiimide
● To have better optical quality of the cells ● Miscellaneous: picric acid

B. Ideal Fixative 1. Simple (only one chemical

● Prevention of autolysis of the cells or tissue present)
● Prevention of decomposition of the tissue by bacteria a. Formaldehyde
○ Most of the fixatives kill a wide spectrum of b. Ethyl alcohol
infectious agents c. Glutaraldehyde
○ Kills viruses (HIV, hepatitis B) but it cannot kill C. Component d. Picric acid\
prions. present e. Osmium tetroxide
■ Prions = misfolded proteins
■ Build up in the brain and occupy space, 2. Compound ( more than
replacing your facial logic leading to one chemical present)
neurologic symptoms etc. a. Bouin’s fluid
■ Prions cause mad cow disease, b. Carnoy’s solution
creutzfeldt-Jakob disease, kuru, and
scrapie. 1. Coagulative: ethyl alcohol,
● Maintaining the volume and shape of the cell as far as picric acid
D. Action on
possible 2. Noncoagulative:
protein formaldehyde, osmium
● Consistently high-quality staining particularly routine stain
tetroxide, glutaraldehyde
such as haematoxylin and eosin stain and Papanicolaou’s
stain
● Rapid action ● Immersion fixation
● Cheap ○ Immerse tissue in the fixative
● Non-toxic ○ 10-20 times the volume
● Coating fixation
C. Changes in Tissue After Fixation ○ Use this in pap smear
● Volume changes ○ Pap smear = take samples of the female patient’s
○ Shrinkage of the volume by formalin (33%). cervix esp the transformation zone
○ Hyperosmolar solution = shrink

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● Freeze drying - frozen section biopsies
○ Same with other types of fixative, but instead of
making cross-linkages between proteins or
removing the water in alcohol
○ It just forces it to be frozen -- if temperature goes
down, all metabolic processes will halt
○ When you force the temperature to go negative to
zero, then all metabolic processes will eventually
stop
■ Inhibits autolysis and putrefaction
■ Because even if there are bacterias
present, they are still bound to the laws
of physics -- if frozen, enzymes will do
stuff; tissues wont decay = tissue will
harden
■ If it's hard, we can cut it to make rapid
slides
○ Very fast than traditional fixation
■ Has a lot of drawbacks:
● A lot of artifacts
● Very hard to make consistent
ribbons
● Chemical properties
○ Aldehyde - most common
○ Cross-linking agents: carbodiimide and
miscellaneous: picric acid
■ Not really encountered here, possible in
higher institutions or abroad
● Component present
○ formaldehyde/ pure formaldehyde
■ Highly doubt that pure formaldehyde is
in HP Labs because it's always
phosphate-buffered
■ Buffer - to make sure that the pH stays
the same; they react to spontaneous
reactions
○ Ethyl alcohol
■ Mostly used blood smears (PBS)
○ Glutaraldehyde and formaldehyde are technically
the same
■ Glutaraldehyde is more expensive
● Less irritation
● Used for electron microscopy
■ Formaldehyde is very irritating to the
nose, skin, and eyes
● At the end of the day, your fixatives only acts on the
proteins
○ Because the proteins are your structural
components (biochemicals) of the cell
○ Inhibit or amplify the proteins/ enzymes
MECHANISM OF FIXATION
● Dehydration and coagulation of protein:
○ Methanol and ethanol (alcohols: OH group)
■ OH groups are hydrophilic (attracts
water)
■ removes attracts water via Hydrogen
bonds
○ Structure of protein
■ Primary protein structure - composed of
sequence of chain of amino acids
■ Secondary protein structure - When
there is folding (due to attraction of side
chains of amino acids)
● either alpha helix or beta
pleated
■ Tertiary protein structure - 3-dimensional
■ Quaternary - more than 1 protein
(cofactors, etc.)
○ Denaturation (dehydrogenation type)
■ Remove liquid pockets
■ In between the strands of helix, there
are water molecules
■ If water is removed, secondary protein
structure is disrupted (up to quaternary)
● They rely on water
■ That's how alcohol kills bacteria, it
coagulates the protein
● Cool effect is caused by water
going to the area
● Alcohol brings water to the
atmosphere = dryness
■ Coagulation of protein can also be
through heating, etc.
○ However, the cell will still show its histologic
structure
● Cross-linking fixatives:
○ Aldehydes - forms methylene bridges between
amino acids leading to a more rigid structure
- Your aldehyde forms methyl linkages in between your
lysine sidechains
- Primary structure is still present
- Secondary structure is also present but there are
alterations
- However, your enzymes still couldn't process what they
need to do
- Proteins are highly fluid. They need to move around, thus
inhibiting them would cause the cell to die
FACTORS AFFECTING FIXATION
● pH of the fixative
○ Neutral pH is preferable
○ pH 6-8 is the best range.
■ Because it is somewhat near to the
physiologic pH
○ High acidity or alkalinity interferes fixation
● Temperature
○ Room temperature suitable for routine work
○ High temperature facilitates fixation
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 ■ In general, if there is a higher
temperature, there will be an increase in
the movement of molecules in the
solution
■ This leads to a faster chemical reaction
○ Low temperature (0-4C) suitable for enzyme
histochemistry
● Duration of fixation
○ Depth of penetration of fixative is directly
proportional to the square root of time of fixation
■ Remember: formalin is a solution
■ How far the formalin is absorbed in the
tissue
○ Formalin fixes 1mm/h
■ Formalin fixation is dependent on how
thick the specimen is
■ Thicker the specimen, the longer the
fixation is
■ Formalin needs to be replaced from time
to time
○ Small tissue: 6h in formalin is optimum
○ Large tissue: 24h is the optimum time
○ Prolonged fixation in aldehyde: inhibition of
enzymatic activity
● Osmolarity of the fixative solution
○ Hypertonic: cell shrinkage
○ Hypotonic: cell swelling
■ Usually seen in your glacial acetic acid
fixatives
○ Best: mild hypertonic (400-450 mOsm)
● Concentration
○ Mild lower concentration with neutral pH is
preferable
○ Very low concentration prolongs the time of
fixation
○ Higher concentration causes rapid fixation with
undesirable effect
● Agitation
○ Agitation increases rate of penetration
○ Rapid agitation: damages delicate tissue
○ Slow gentle agitation preferable
○ How to make the chemical reaction faster?
■ Increase the amount or concentration of
components
■ Use heat (ex. Preparing coffee)
■ Agitation
● More clash of molecules =
more chemical reactions
● Disadvantages
○ Slow fixation.
○ Formalin reaction is reversible, and it can be
removed by washing.
■ If washed, methylene bridges will break
and malata.
○ Fails to preserve acid mucopolysaccharides.
○ Not good for staining of fat and enzymes.
○ Highly vascular tissue may have dark-brown
granules.
■ Because it oxidizes and lyses your
blood.
○ Exposure to the skin may cause dermatitis.
○ Chronic inhalation may cause bronchitis.
Glutaraldehyde
● Glutaraldehyde is used as a fixative for electron microscopy
because it fixes and preserves the ultrastructure. The
fixation occurs due to the extensive cross-linking of the
proteins.
● The penetration power of glutaraldehyde is poor and
therefore only a small piece of tissue should be used for
fixation.
● Glutaraldehyde does not react with lipid or carbohydrate,
and therefore it should be used in combination with the
other fixative.
○ Advantages
■ Better fixation of ultrastructure.
■ Less cell shrinkage.
■ Preservation of protein is better.
■ Good cross-linking with collagen.
■ Less irritating.
○ Disadvantages;
■ Poor penetration and tissue should be
less than 0.5 mm thick.
■ Less stable compound.
■ No lipid fixation.
■ Glutaraldehyde polymerizes above pH
7.5.
■ Costly

COMMONLY USED FIXATIVES IN THE LABORATORY

● FORMALDEHYDE
○ Formaldehyde in its pure form is a gas
○ Commercially available as 40% concentration
(considered as 100% formalin)
○ 10% of this formalin is used to make neutral
buffered formalin
○ Mechanism:
■ It reacts with various side chains of the
protein and forms hydroxymethyl side
groups that subsequently cross-link to
form a methylene bridge. Subsequent
intermolecular and intramolecular
cross-linking of the molecules occurs
and ultimately produces an insoluble
product
○ Rate of penetration
■ Formalin penetrates approximately
1mm/h
○ Volume of formalin
■ The amount of formalin should be 20
times the volume of tissue
○ Advantages ● Phosphate buffer- commonly used formalin, to maintain it
■ High penetration rate into the ideal ph (6-8)
■ Well-preserved cell morphology ● Picric acid- explosive
■ Cheap ○ Should be placed in amber colored glass bottle
■ Stable
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 ● Glacial acetic acid- makapataba sa cells because it is
hypoosmolar and might produce cellular swelling but it is TROUBLE SHOOTING IN FIXATION
counteracted by mercuric chloride.
Problems Cause Remedies

Nuclear margin is Incomplete fixation - check the

indistinct and nuclei concentration of
are fuzzy with formalin
bubbling -keep more time in
formalin fixation

Tissue shrinkage -poor fixation -poor fixation time

with large artifactual -prolonged fixation -check fixative
spaces concentration
-immediately fix the
tissue after biopsy

Insoluble Formalin pigment Use buffered

brownish-black due to acid formalin
granular pigment formaldehyde
hematin formation
by reaction with
blood

Intraepithelial cleft Formalin may Keep formalin in

formation evaporate, and closely capped
calcium carbonate bottle
is precipitated.

Karyorrhexis-
● Nuclear margin is indistinct and fuzzy
● Fragmentation of nucleus due to autolysis
● wala sha na fixed immediately, naa pay metabolic
processes na ga continue→ decrease in ATP→ cell
undergo apoptosis

Fixation Artefact
● Formalin pigment: Insoluble brownish black granular
retractile birefringent pigment due to reaction of formalin
with haemoglobin derivatives.
● Mercury pigments: Dark-brown irregular deposit.
● Fuzzy staining: Due to improper fixation.
○ Cold ischemia time
■ Affects lots of test including
immunohistochemistry
● Prolonged fixation: Shrinkage of the tissue causes tissue
separation and empty spaces.
● Dichromate deposit: This deposit may occur after
dichromate fixation if the tissue is not washed properly.
Formalin Pigment
● Colour: Brownish black.
● Nature: Granular birefringent refractile.
● Position: Extracellular.
● Mechanism of formation: Formic acid reacts with
haemoglobin derivatives of the blood and produces acid
formaldehyde hematin.
○ Formation of formic acid- because formaldehyde
will eventually become formic acid if your solution
is not buffered.
● How to avoid: Use buffered formalin.
● How to remove: Treat with 1.8% picric acid in absolute ethyl
alcohol for 15 min.
● PICRIC ACID IS AN EXPLOSIVE.