GENERAL PATHOLOGY LECTURE (MIDTERMS)

GROUP 1 HANDOUT
FIXATION
The process at which it involves fixing
and preserving fresh tissue for
examination.
The shape, structure, intercellular
relationship and chemical constituents
of tissues are preserved by preventing
degeneration, putrefaction,
decomposition and distortion of tissues
after death.
It can be done by chemical or physical
methods
In physical fixation heat,
freezing and desiccation are
used.
In chemical fixation This is
considered the primary method
of fixation. The tissues are
usually immersed in solutions
of stabilizing or cross-linking
agents called fixatives. These
chemical reagents may be
classified as additive and nonadditive or coagulant and noncoagulant.
NON ADDITIVE FIXATIVE
Acts on the tissue without chemically
combining with the tissue.
Examples are alcohol and acetone.
ADDITIVE
They chemically link or bind to the
tissue and change it.
The disruption enables the protein to
combine w/ a fixative molecule, and the
protein then becomes insoluble.

Examples are Mercuric Chloride,

Chromium Trioxide, Zinc Sulfate,
Chloride

COAGULANT
Will allow the solutions to penetrate
into the interior of the tissue very
easily.
Examples are Zinc salts, Acetone, Picric
Acid, Alcohols (methyl/ethyl), Mercuric
Chloride, Cupric Sulfate
PURPOSE/GOAL
To preserve the morphologic and
chemical integrity of the cell in as lifelike matter as possible.
To harden and protect the tissue from
the trauma of further handling, so that
it is easier to cut during gross
examination.
MAIN FACTORS INVOLVED IN
FIXATION
1. pH
2. Temperature
3. Size and thickness of section
4. Osmolality
5. Concentration
6. Duration of Fixation
7. Penetration
PRACTICAL CONSIDERATION OF
FIXATION
1. Speed- this is done to prevent
autolysis and putrefaction
2. Penetration- formalin diffuses into
the tissue at the rate of approximately
1mm per hour and slows down as it
goes deeper into the tissue
3. Volume- Traditionally: amount of
fixative used has been 10-25 times the

volume of tissue. Recently: maximum

effectiveness of fixation noted to be 20
times the issue volume. 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).
4. Duration of Fixation- takes longer to
fix than others depending on their
structure.
CHARACTERISTICS OF A GOOD
FIXATIVE
1. it must be cheap
2. it must be stable
3. it must be safe to handle
4. it must kill the cell quickly thereby
producing minimum distortion of cell
constituents.
5. it must inhibit bacterial
decomposition and autolysis.
6. it must produce minimum shrinkage
of tissues.
7. it must permit rapid and even
penetration of tissues.
8. it must harden tissues thereby
making the cutting of sections easier.
9. it must be isotonic, causing minimal
physical and chemical alteration of the
cells and their constituents.
10. it must make cellular components
insoluble to hypotonic solutions and
render them insensitive to subsequent
processing
11. it must permit the subsequent
application of many staining
procedures to facilitate easier and more
profitable examination
EFFECTS OF FIXATIVES
1. They harden and soften friable
tissues and make the handling and

cutting of tissues easier. This is usually

accelerated by the action of alcohol
during the dehydration process.
2. They make the cells resistant to
damage and distortion caused by
hypotonic and hypertonic solutions
used during tissue processing.
3. They inhibit bacterial decomposition
4. They increase the optical
differentiation of cells and tissue
components thereby rendering them
more readily visible during
examinations
5. They act as a mordant or
accentuators to promote and hasten
staining or they may inhibit certain
dyes in favor of another.
6. They reduce the risk of infections
during handling and actual processing
of tissues.

PRINCIPLES AND PRECAUTIONS

IN HANDLING FIXATION OF
SPECIMENS IN GENERAL
1. Autopsy materials should be fixed as
soon as possible after death to prevent
decomposition and autolysis.
2. Surgical specimens should be fixed as
soon as possible after removal.
3. If specimen is placed in normal saline
solution, autolysis may occur.
4. All tissue specimens must be
properly labeled and identified.
5. Slow freezing of unfixed tissue near
0C must be avoided.
6. Tissue slices should be taken at right
angles to the surface of the organ and
should be sufficiently deep to show
normal anatomic components.
7. Tissues should NOT be more than
5mm.

 8. Thin sections allow complete

penetration by fixative in a short time.
9. Purulent materials, exudates, or
transudates should be marked and
kept for possible cultures, smears and
other bacteriologic examination.
10. The amount of fixative must be
adequate
11. For prolonged fixation, volume of
fixing fluid should NOT be less than 50100 times that of the tissue.
12. Contamination of fixed tissue with
precipitates should be avoided.
13. Fixed tissues must be washed
thoroughly to remove salts and/or
pigments before staining.
14. Low temperature retards fixation
but prevents autolysis.
15. The required period for fixation
should not be exceeded.
16. There must always be an adequate
supply of fixatives at all times.
17. Drying should be avoided.
18. Hollow organs should be packed
with cotton soaked in fixative or
completely opened before being
immersed in fixing solution.
19. Air-filled lungs may float on fixative.
20. Human brains may be suspended
by a cord tied under the circle of Willis
to prevent flattening, and may undergo
intravascular perfusion.
21. Eyes should NOT be dissected
before they are fixed.
22. Frozen sections may lead to
formation of ice crystal artifacts.
23. When fixing muscles, the fresh
biopsy material may be stretched or
laid flat in a moist filter paper before
suspending in fixative.
24. Water should NOT be used for
glycogen-containing tissues.

25. To assure rapid access of the fixative

to all parts of the tissue, the tissue may
be minced.
26. Hard tissues may be washed out in
running water overnight and immersed
in 4% aqueous phenol solution for 1-3
days.

Most Commonly used fixatives

I.
FORMALIN
II.
ALCOHOL
I Formaldehyde (FORMALIN)
It is a gas produced by the oxidation of
methyl alcohol, and is soluble in water
to extent of 37-40% weight in volume.
Mechanism: It forms cross links
between amino acids of proteins
thereby making them insoluble.
Fixation time: 24 hours
10% formalin
Advantages:
1. cheap, readily available, easy to prepare, and
relatively stable
2. compatible w/ many stains
3. does not over harden tissues

4. penetrates tissues well

5. preserves fat, mucin, proteins and glycogen.
6. Does not make tissues brittle
7. Restore natural tissue colors
8. Does not require washing out
Disadvantages:
1. Irritating fumes to nose and eyes.
2. Solution is irritating to skin
3. May produce considerable shrinkage of
tissues
4. Does not adequately harden some
cytoplasmic structures for paraffin embedding.
5. Prolonged fixation may yield bleaching the
specimen and loss of natural tissue color, and
also dispersion of fat from tissue to fluid
% Absolute Formalin
- It is pure stock 40% formalin which is
unsatisfactory for routine fixation.
-It has high formaldehyde concentrates
which tends to overharden the outer layer of
tissue and affect staining adversely.
-Fixation time: 24 hours
10% Neutral Buffered Formalin
- recommended for preservation and
storage of surgical, post-mortem, and research
specimen.
-fixation time: 4-24 hours
-phosphate buffered formalin
10% Neutral Buffered Formalin
Advantages:
1. Prevents precipitation of acid formalin
pigments on post-mortem tissues
2. Best fixative for tissues containing iron
pigments and for elastic fibers
Disadvantages:
1. It is longer to prepare
2. Positivity of mucin to PAS is reduced
3. May produce gradual loss of basophilic
staining of cells

4. Reduced reactivity of myelin to

Weigerts Iron Hematoxylin stain
5. It is inert towards lipids.
II ALCOHOL
Mechanism: It rapidly denatures and
precipitates proteins by destroying hydrogen
and other bonds.
It must be used in concentrations ranging from
70-100%
Ideal for small tissue fragments
Not used for lipid studies
Preserves glycogen but cause glycogen
polarization
100% Methyl Alcohol
- excellent for fixing dry and wet
smears, blood smears and bone marrow tissue
-Penetration is slow
-may overharden if left for more than
48 hours
Ethyl Alcohol
-used at concentrations of 70-100%
-< 70-100% hemolysis and inadequate
WBC preservation
-more frequently incorporated into
compound fixative for better results.
-preserves but does not fix glycogen
-fixes blood, tissue films and smears
-used for histochemistry and enzyme
studies
-fixes tissue pigments
-cause glycogen polarization
-produce considerable hardening and
shrinkage of tissues
-strong reducing agent.
OTHER FIXATIVES
Formal-Corrosive (Formal-sublimate)
-Formol-mercuric chloride solution is
recommended for routine post-mortem issues.

 Alcoholic Formalin (Gendres) Fixative

-fixation is faster
-Better preservation of glycogen
-Useful for sputum

acid penetrates thoroughly and rapidly but

lyses red blood cells.

Glutaraldehyde-made up of two formaldehyde

residues, linked by three carbon chains.
-It acts in a manner similar to
formaldehyde and is sometimes utilized for
routine microscopic work.

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
-is the most common fixative in the
past, frequently used in saturated aqueous
solutions of 5-7%.
-These penetrate rapidly and
precipitate all proteins.
-It should also be noted that mercuric
salts are highly toxic and must not be disposed
into sewerage systems

Acetone
-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.

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.

10% Formol Saline

-Slow fixative
-simple microanatomical fixative made
up of 10% saturated formaldehyde and sodium
chloride.
-recommended for fixation of central
nervous tissues and general post-mortem
tissues for histochemical examination.
-fixation time: 24 hours @ 35C & 48
hours @ 20-25C

Chromic Acid
-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.
Acetic Acid
-Acetic acid is never used alone but is
often combined with other fixatives that cause
shrinkage such as ethanol and methanol. Acetic

Zenkers Fluid
-recommended for fixing small pieces
of liver, spleen, connective tissue fibers and
nuclei. Fixation time is 12-24 hours.
Hellys Solution
-It is mixed with small amount of
formalin and preserves red blood cells. Fixation
time: 12-24 hours

-recommended for nuclear

preparations of such sections. Fixing time: 2448 hours
Heidenhains Susa Solution
- recommended mainly for tumor
bioppsies especially of the skin. It is an
excellent cytologic fixative.
Fixation time: 3-12 hours
Lead fixative
-recommended for acid
mucopolysaccharides and connective tissue
mucin
Regauds (Mollers) Fluid
-recommended for demonstration of
chromatin, mitochondria, mitotic figures Golgi
bodies, RBC, and colloid-containing tissues.
Fixation time: 12-48 hours
Orths Fluid
-recommended for the study of early
degenerative processes and tissue necrosis.
And it also demonstrate Rickettsia and other
bacteria. Fixation time: 36-72 hours
Bouins Solution
-recommended for fixation of embryos.
Fixation time: 6-24 hours
Carnoys Fluid
-recommended for fixing
chromosomes, lymph glands and urgent
biopsies. Fixation time: 1-3 hours
Newcomers fluid
-recommended for fixing
mucoplysaccharides and nuclear proteins
Flemmings Solution

B5
-commonly used for bone marrow
biopsies.
FIXATIVES IN ORDER OF SPEED
OF PENETRATION, FASTEST TO
SLOWEST
1. Formalin
2. Acetic Acid
3. Mercuric Chloride
4. Methyl Alcohol
5. Osmium Tetroxide
6. Picric Acid
HEAT FIXATION
This process involves thermal
coagulation of tissue proteins for rapid
diagnosis, usually employed for frozen
tissue sections and preparation of
bacteriologic smears.