Fixation is a procedure
adopted to kill, harden and preserve materials for microscopic study, by means
of a substance known as fixative. The shape, structure, intercellular
relationship and chemical constituents of tissues are preserved by preventing
degeneration, putrefaction, decomposition and distortion of tissues after
death, consequent to cutting and removal from the body.
Effects
of fixative in general:
1. They harden soft and friable
tissues and make the handling and cutting of sections 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 solution 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 examination
5. They act as mordants or
accentuators to promote and hasten staining, or they may inhibit certain dyes
in favor of another (e.g. formaldehyde intensifies while osmium tetroxide
inhibits hematoxylin).
6. They reduce the risk of infection
during handling and actual processing of tissues.
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 thereby
producing minimum distortion of cell constituents.
5. It must inhibit bacterial
decomposition and autolysis.
6. It must produce minimum shrinkage
of tissue.
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.
This is not, however, a strict rule
since there are some hypotonic solutions (i.e. Glacial Acetic Acid) producing
tissue swelling, which are being used in conjunction with hypertonic solutions
(e.g. Picric Acid) causing shrinkage of cells, to produce a compound which
would give an optimal effect on the tissue structure.
10. It
must make the 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.
Types
of Fixatives:
I. According to
composition
A. Simple
fixatives
– made up of only one component susbstance
1. Aldehydes
Aldehyde
fixatives are satisfactory for routine paraffin sections, for electron
microscopy and when histochemical and enzyme studies are indicated.
a. Formaldehyde (Formalin)
This is a gas produced by the oxidation
of methyl alcohol, and is soluble in water to the extent of 37 – 40% weight in
volume. Pure stock solution of 40% formalin is unsatisfactory for routine
fixation since high formaldehyde concentrates tend to over harden the outer
layer of the tissue and affect staining adversely. Therefore, it must be
diluted 1:10 or 1:20 to make a 10% or 5% solution, or combined with another
fixative to form a compound solution. Usual fixation time is 24 hours.
Advantages:
(1) It
is cheap, readily available, easy to prepare, and relatively stable, especially
if stored in buffered solution.
(2) It
is compatible with many stains, and therefore can be used depending upon the
need of the tissues.
(3) It
does not over harden tissues, even with prolonged periods of fixation, as long
as solutions are regularly changed.
(4) It
penetrates tissues well.
(5) It
preserves fat and mucin, making them resistant to subsequent treatment with fat
solvents, thereby allowing tissue enzymes to be studied.
(6) It
preserves glycogen.
(7) It
preserves but does not precipitate proteins, thereby, allowing tissue enzymes
to be studied.
(8) It
does not make tissues brittle, and is therefore recommend for nervous tissue
preservation.
(9) It
allows natural tissue colors to be restored after fixation by immersing
formalin – fixed tissues in 70% alcohol for one hour, and is therefore
recommended for colored tissue photographies.
(10)
It allows frozen sections to be prepared easily.
(11)
It does not require washing out, unless tissues have stayed in formalin for
excessively long period of time.
Disadvantages:
(1) Fumes
are irritating to the nose and eyes may cause sinusitis, allergic rhinitis or
excessive lacrimation.
(2) The
solution is irritating to the skin and may cause allergic dermatitis on
prolonged contact.
(3) It
may produce considerable shrinkage of tissues.
(4) It
is a soft fixative and does not harden some cytoplasmic structures adequately
enough for paraffin embedding.
(5) If
unbuffered:
(a) Formalin
reduces both basophilic and eosinophilic staining of cells, thereby reducing
the quality of routine cytologic staining. Acidity of formic acid may, however,
be used to an advantage when applying the Silver Impregnation Technique of
staining.
(b) It
forms abundant brown pigment granules on blood–containing tissues.
e.g. spleen, due
to blackening of hemoglobin
(6) Prolonged
fixation may produce:
(a) Bleaching
of the specimen and loss of natural tissue colors.
(b) Dispersal
of fate from the tissues into the fluid.
Precaution:
(1) Prolonged
storage of formaldehyde especially at very low temperature, may induce
precipitation of white formaldehyde deposits and produce turbidity although
this, in itself, does not impair the fixing property of the solution.
Precipitates may be removed by filtration or by addition of 10% methanol.
Methanol added as a preservative to
formaldehyde will prevent its decomposition to formic acid or precipitation to
paraformaldehyde, but it serves to denature protein, thereby rendering formalin
unsuitable as a fixative for electron microscopy.
(2) Room
should be properly ventilated with adequate windows and preferably with an
exhaust fan to prevent inhalation of fumes and consequent injury to the eyes
and nose.
(3) Dermatitis
may be avoided by the use of rubber gloves when handling specimens fixed in
formalin.
(4) Bleaching
of tissues may be prevented by changing the fluid fixative every three months.
(5) Natural
tissue color may be restored by immersing tissues in 70% alcohol after
fixation.
(6) If
fatty tissues are to be stored for a long time, cadmium or cobalt salts are
added to prevent dispersion of fat out into the fluid.
(7) Acid
reaction due to formic acid formation can be buffered or neutralized by adding
magnesium carbonate or calcium carbonate to 10 – 15% formalin. This should be
done on a wide mouth bottle to prevent violent explosion due to insufficient
gas space for CO2 release.
(8) To
improve staining and produce firmer and harder consistency, tissues fixed in
formalin for 1 – 2 hours may be placed again in Helley’s fluid for 4 – 6 hours
or in formol–sublimate for 4 – 16 hours (secondary fixation).
(9) If
post–fixed in osmic acid, the tissue must be washed in demineralized water to
prevent hypotonicity and bleaching.
b. Glutaraldehyde – is made up of
two formaldehyde residues, linked by three carbon chains, utilized for routine
light microscopic work and also satisfactory for electron microscopy.
A 2.5% solution is used for small tissue
fragments and needle biopsies fixed in 2 – 4 hours at room temperature. A 4%
solution is recommended for larger tissues less than 4 mm thick, fixed in 6 – 8
hours up to 24 hours.
Advantages of
glutaraldehyde over formalin:
(1) It
has a more stable effect on tissues, giving firmer texture with better tissue
sections, especially of central nervous tissues.
(2) It
preserves plasma proteins better.
(3) It
produces less tissue shrinkage.
(4) It
preserves cellular structures better, hence, is recommended for enzyme
histochemistry and electron microscopy.
(5) It
is more pleasant and less irritating to the nose.
(6) It
does not cause dermatitis.
Disadvantages:
(1) It
is more expensive
(2) It
is less stable
(3) It
penetrates tissues more slowly.
(4) It
reduces PAS positivity of reactive mucin. This may be prevented by immersing
glutaraldehyde – fixed tissues in a mixture of concentrated glacial acetic acid
and aniline oil.
2. Metallic
fixatives
a. Mercuric
chloride
– is the most common fixative, used in saturated aqueous solutions of 5 – 7%;
it is included in many compound fixatives.
Advantages:
(1) It
penetrates and hardens tissues rapidly and well.
(2) Nuclear
components are shown in fine detail.
(3) It
precipitates all proteins.
(4) It
has a greater affinity to acid dyes and is preferred in lieu of formaldehyde
for cytoplasmic staining.
(5) Trichome
staining is excellent.
(6) It
is the routine fixative of choice for preservation of cell detail in tissue
photography.
(7) It
permits brilliant metachromatic staining of cells.
(8) It
is recommended for renal tissues, fibrin, connective tissues and muscles.
Disadvantages:
(1) It
causes marked shrinkage of cells (this may be counteracted by addition of acid)
(2) It
rapidly hardens the outer layer of the tissue with incomplete fixation of the
center, therefore, thin sections should be made.
(3) Penetration
beyond the first 2 – 3 millimeters is slow, hence, not more than 5 mm thickness
of tissues should be used.
(4) If
left in fixative for more than 1 – 2 days, the tissue becomes unduly hard and
brittle.
(5) It
prevents adequate freezing of fatty tissues and makes cutting of frozen tissues
difficult.
(6) It
causes considerable lysis of red blood cells and removes much iron from
hemosiderin.
(7) It
is inert to fats and lipids.
(8) It
leads to the formation of black granular deposits in the tissues.
(9) It
reduces the amount of demonstrable glycogen.
(10)
Compound solutions containing Mercuric chloride deteriorate rapidly upon
addition of Glacial Acetic Acid to Formalin
(11)
It is extremely corrosive to metals.
Precaution
(1) Black
deposits may be removed by adding saturated iodine solution in 96% alcohol, the
iodine being decolorized with absolute alcohol in the subsequent stages of
dehydration.
(2) Compound
solutions must always be freshly prepared.
(3) The
use of metals caps to cover the bottles containing fixative should be avoided.
(4) Contact
of mercuric fixatives with personal jewelry should be avoided.
Just before use,
Glacial Acetic Acid may be added to form Zenker’s solution or strong
Formaldehyde may be added to form Zenker – Formol solution (Helly’s).
b. Chromate
fixative
(1) Potassium
dichromate
– is used in 3% aqueous solution.
(a) It
fixes but does not precipitate cytoplasmic structures.
(b) It
preserves lipids.
(c) It
preserves mitochondria (if used in pH 4.5 – 5.2, mitochondria are fixed; if
solution becomes acidified, cytoplasm, chromatin bodies and chromosomes are
fixed but mitochondria are destroyed.)
(2) Chromic
acid
– is used in 1 – 2% aqueous solution, usually as a constituent of a compound
fixative. It precipitates all proteins and adequately preserves carbohydrates.
It is a strong oxidizing agent; hence, a strong reducing agent (e.g.
formaldehyde) must be added to chrome–staining fixative before use in order to
prevent counteracting effects and consequent decomposition of solution upon
prolonged standing.
c. Lead
fixatives
Lead fixatives are used in 4% aqueous
solution of basic lead acetate
Advantage:
(1) It
is recommended for acid mucopolysaccharide.
(2) It
fixes connective tissue mucin
Disadvantage:
It takes up CO2 to form
insoluble lead carbonate especially on prolonged standing. This may be removed
by filtration or by adding acetic acid drop by drop to lower the pH and
dissolve the residue.
3. Picric acid
Picric acid is normally used in strong
saturated aqueous solution (approximately 1%)
Advantages:
a. It is an excellent fixative for
glycogen demonstration
b. It penetrates tissues well and
fixes small tissues rapidly
c. The yellow stain taken in by
tissue prevents small fragments from being overlooked
d. It allows brilliant staining with
the Trichome method
e. It is suitable for aniline stains
(Mallory’s Heidenhain’s or Mason’s method
f. It precipitates all proteins
g. It is stable
Disadvantages:
a. It causes RBC hemolysis and
reduces the amount of demonstrable ferric iron in tissues.
b. It is not suitable for frozen
sections because it causes frozen sections to crumble when cut.
c. Prolonged fixation makes tissues
hard, brittle and difficult to section. Tissues should not be allowed to remain
in the fluid for more than 12 – 24 hours (depending on size).
d. Picrates are formed upon protein;
precipitates are soluble in water, hence, tissues must be first rendered
insoluble by direct immersion in 70% ethyl alcohol.
Picric acid fixed tissues must never be
washed in water before dehydration.
e. Picric acid will produce
excessive staining of tissues. To remove the yellow color, tissues may be
placed in 70% ethyl alcohol followed by 5% sodium thiosulfate and then washed
in running water.
f. Picric acid is highly explosive
when dry, and therefore must be kept moist with distilled water or saturated
alcohol at 0.5 to 1% concentration during storage.
g. It alters and dissolves lipids.
h. It interferes with azure eosin
method of staining; hence, tissues should be thoroughly washed with alcohol.
4. Acetic acid
Acetic acid is
normally used in conjunction with other fixatives to form a compound solution.
It solidifies at 17oC, hence, the name Glacial Acetic Acid.
Advantages:
a. It fixes and precipitates
nucleoproteins.
b. It precipitates chromosomes and
chromatin materials; hence, is very useful in the study of nuclear components
of the cell. In fact, it is an essential constituent of most compound nuclear
fixatives.
c. It causes tissues (especially
those containing collagen) to swell. This property is used in certain compound
fixatives to counteract the shrinkage produced by other components (e.g.
mercury)
Disadvantages:
a. When combined with potassium
dichromate, the lipid – fixing property of the latter is destroyed (e.g.
Zenker’s fluid)
b. It is contraindicated for
cytoplasmic fixation since it destroys mitochondria and Golgi elements of
cells.
5. Acetone
Used at ice cold
temperature ranging from –5oC to 4oC.
Advantages:
a. It is recommended for the study
of water diffusible enzymes especially phosphatases and lipases.
b. It is used in fixing brain
tissues of diagnosis of rabies.
c. It is used as a solvent for
certain metallic salt to be used in freeze substitution technique for tissue
blocks.
Disadvantages:
a. It produces inevitable shrinkage
and distortion.
b. It dissolves fat.
c. It preserves glycogen poorly.
d. It evaporates rapidly.
6. Alcohol
Alcohol rapidly
denatures and precipitates proteins by destroying hydrogen and other bonds. It
must be used in concentrations ranging from 70 to 100% because less
concentrated solutions will produce lysis of cells.
Advantages:
a. It is ideal for small tissue
fragments.
b. It may be used both as a fixative
and dehydrating agent.
c. It is excellent for glycogen
preservation.
d. It preserves nuclear stains.
Disadvantages:
a. It causes RBC hemolysis
b. It dissolves fats and lipids; as
a general rule, alcohol containing fixatives are contraindicated when lipids
are to be studied.
7. Osmium tetroxide
This is a pale
yellow powder which dissolves in water (up to 6% at 20oC) to form a
strong oxidizing solution.
Advantages:
a. It fixes conjugated fats and
lipids permanently by making them insoluble during subsequent treatment with
alcohol and xylene.
Fats from hydrated osmium dioxide, are
stained black and therefore are easier to identify.
b. It preserves cytoplasmic
structures well, e.g. Golgi bodies and mitochondria.
c. It fixes myelin and peripheral
nerves well, hence, is used extensively for neurological tissues.
d. It produces brilliant nuclear
staining with safranin
e. It adequately fixes materials for
ultrathin sectioning in electron microscopy, since it rapidly fixes small pieces
of tissues and aids in their staining.
f. It precipitates and gel protein.
g. It shows uniformly granular
nuclei with clear cytoplasmic background.
h. Some tissues (e.g. adrenal
glands) are better fixed in vapor form of osmium tetroxide. This eliminates “washing
out” of the fixed tissue.
Disadvantages:
a. It is very expensive.
b. It is a poor penetrating agent,
suitable only for small pieces of tissues.
c. It is readily reduced by contact
with organic matter and exposure to sunlight, forming a black precipitate,
which settles at the bottom of the container.
d. Prolonged exposure to acid vapor
can irritate the eye, producing conjunctivitis, or cause the deposition of
black osmic oxide in the cornea, producing blindness.
e. It inhibits hematoxylin and makes
counterstaining difficult.
f. It is extremely volatile.
Precaution:
a. It should be kept in a
dark–colored, chemically clean bottle to prevent evaporation and reduction by
sunlight or organic matter.
b. Eyes and skin may be protected by
using a fume hood or wearing protective plastic masks or gloves while using
osmium tetroxide.
c. Addition of saturated aqueous
mercuric chloride solution (0.5 to 1ml / 100 ml of stock solution) will prevent
its reduction with formation of black deposits.
d. Black osmic oxide crystals may be
dissolved in cold water.
e. Osmic acid – fixed tissues must
be washed in running water for at least 24 hours to prevent formation of
artifacts.
f. To prevent contact of tissues
with black precipitate formed in the bottom of the jar, the tissue may be
wrapped in cotton gauze and suspended in the fluid by means of a thread.
8. Heat
This procedure
involves thermal coagulation of tissue proteins for rapid diagnosis, usually
employed for frozen tissue sections and preparation of bacteriologic smears.
Advantages:
a. Fixation is better
b. It preserves nuclear and
cytoplasmic detail
c. It is suitable for frozen tissue
preparation.
Disadvantages:
a. It produces considerable tissue
shrinkage and distortion
b. It destroys RBC
c. It dissolves starch and glycogen.
B. Compound
fixatives
– are made up of two or more fixatives which have been added together to obtain
the optimal combined effect of their individual actions upon the cells and
tissue constituents.
II. According to
action
A. Microanatomical
fixatives
Are those which permit the general
microscopic study of tissue structures without altering the structural pattern
and normal intercellular relationship of the tissues in question.
1. 10% formol
saline
– is a simple microanatomical fixative made up of saturated formaldehyde (40%
grams by weight volume) diluted to 10% with sodium chloride; it is recommended
for fixation of central nervous tissues and general post–mortem tissues for
histochemical examination.
Fixation time: 24 hours at 35oC
48 hours at 20 –
25oC
Advantages:
a. It
penetrates and fixes tissue evenly.
b. It
preserves microanatomic and cytologic details with minimum shrinkage and
distortion.
c. Large
specimen may be fixed for a long time provided that the solution is changed
every three months.
d. It
preserves enzymes and nucleoproteins.
e. It
demonstrates fats and mucin.
f. It does not over harden tissue,
thereby facilitating dissection of the specimen.
g. It
is ideal for most staining techniques, including Silver Impregnation.
h. It
allows natural tissue color to be restored upon immersion in 70% alcohol.
Disadvantages:
Similar to
formaldehyde with the following addition:
a. It is a slow fixative, period of
fixation is required to be 24 hours or longer.
b. Formol–saline fixed tissues tend
to shrink during alcohol dehydration; this may be reduced by secondary
fixation.
c. Metachromatic reaction of amyloid
is reduced.
d. Acid dye stains less brightly
than when fixed with mercuric chloride
2. 10% Neutral
Buffered Formalin
– is recommended for preservation and storage of surgical, post–mortem and
research specimens.
Fixation
time: 4 – 24 hours
Advantages:
Are similar to
formol–saline with the following addition:
a. It prevents precipitation of acid
formalin pigments on post–mortem tissues.
b. It is the best fixative for
tissues containing iron pigments and for elastic fiber which do not stain well
after Susa, Zenker or chromate fixation.
Disadvantages:
a. It is longer to prepare; hence,
is time consuming
b. Positivity of mucin to PAS
reduced.
c. It may produce gradual loss in
basophilic staining.
d. Reactivity of myelin to Weigert’s
Iron Hematoxylin Stain is reduced.
e. It is inert towards lipids,
especially neutral fats and phospholipids.
3. Heidenhain’s
Susa
– recommended mainly for tumor biopsies especially of the skin; it is an
excellent cytologic fixative.
Fixation
time: 3 – 12 hours
Advantages:
a. It penetrates and fixes tissues
rapidly and evenly.
b. It produces minimum shrinkage and
hardening of tissues due to the counter balance of the swelling effects of
acids and the shrinkage effect of mercury.
c. It permits most staining
procedures to be done, including Silver Impregnation, producing brilliant
result with sharp nuclear and cytoplasmic details.
d. It permits easier sectioning of
large blocks of fibrous connective tissues.
e. Susa–fixed tissues may be
transferred directly to 95% alcohol or absolute alcohol, thereby reducing
processing time.
Disadvantages:
a. Prolonged fixation of thick
materials may produce considerable shrinkage, hardening and bleaching; hence,
tissues should not be more than 1 cm thick.
b. RBC preservation is poor.
c. Some cytoplasmic granules are
dissolved.
d. Mercuric chloride deposits tend
to form on tissues; these may be removed by immersion of tissues in alcohol
iodine solution.
e. Weigert’s method of staining
elastic fibers is not possible in susa–fixed tissues.
Precaution:
a. After using Heidenhain’s Susa
fixative, the tissue should be transferred directly to a high grade alcohol,
e.g. 96% or Absolute Alcohol, to avoid undue swelling of tissues caused by
treatment with low–grade alcohol or water.
4. Formol Sublimate
(Formol Corrosive)
– formol–mercuric chloride solution is recommended for routine post–mortem
tissues.
Fixation time: 3 – 24 hours
Advantages:
a. It penetrates small pieces of
tissues rapidly
b. It produces minimum shrinkage and
hardening
c. It is excellent for many staining
procedures including Silver Reticulum methods.
d. It brightens cytoplasmic and metachromatic
stains better than with formalin alone.
e. Cytological structures and blood
cells are well–preserved.
f. There is no need for
“washing–out”; tissues can be transferred directly from fixative to alcohol.
g. It fixes lipids, especially
neutral fats and phospholipids
Disadvantages:
a. Penetration is slow; hence,
tissue sections should not be more than 1cm thick.
b. It forms mercuric chloride
deposits.
c. It does not allow frozen tissue
sections to be made.
d. It inhibits the determination of
the extent of tissue decalcification.
5. Zenker’s
solution
– is made up of Mercuric Chloride stock solution to which Glacial Acetic acid
has been added just before its use. It is recommended for fixing small pieces
of liver, spleen, connective tissue fibers and nuclei.
Fixation
time: 12 – 24 hours
Advantages:
a. It produces a fairly rapid and
even fixation of tissues.
b. Stock solutions keep well without
disintegration.
c. It is recommended for Trichome
staining.
d. It permits brilliant staining of
nuclear and connective tissue fibers.
e. It is compatible with most
stains.
f. It may act as a mordant to make
certain special staining reactions possible.
Disadvantage:
a. Penetration is poor.
b. It is not stable after addition
of Acetic acid.
c. Prolonged fixation (for more than
24 hours) will make tissues brittle and hard.
d. It causes lysis of red blood
cells and removes iron from hemosiderin.
e. It does not permit cutting of
frozen sections.
f. It has the tendency to form
mercuric pigment deposits or precipitates.
g. Insufficient washing may inhibit
or interfere with good cellular staining.
Precaution:
a. Do not let tissues stay in
solution for more than 24 hours.
b. Solutions must always be freshly
prepared.
c. Tissues must be washed out
thoroughly in running water to permit good staining.
d. Mercuric deposits may be removed
by immersing tissues in alcoholic iodine solution.
e. Tissues should be cut thin (2 – 3
mm) and hollow organs should be opened to promote complete penetration and
fixation.
6. Zenker–Formol
(Helly’s solution)
Fixation
time: 12 – 24 hours
Advantages:
a. It is an excellent microanatomic
fixative for pituitary gland, bone marrow and blood containing organs such as
spleen and liver.
b. It penetrates and fixes and
tissues well.
c. Nuclear fixation and staining is
better than Zenker’s.
d. It preserves cytoplasmic granules
well.
Disadvantages:
The disadvantages of Helly’s solution
are similar to Zenker’s except that brown pigments are produced if tissues
(especially blood–containing organs) are allowed to stay in the fixative for
more than 24 hours due to RBC lysis. This may be removed by immersing the
tissue in saturated alcoholic picric acid or sodium hydroxide.
7. Bouin’s solution is recommended
for fixation of embryos
Fixation
time: 6 – 24 hours
Advantages:
a. It produces minimal distortion of
micro–anatomical structures.
b. Yellow stain is useful when
handling fragmentary biopsies.
c. It permits brilliant staining of
tissues.
d. It preserves glycogen.
e. It does not need “washing out.”
Disadvantages:
a. It penetrates poorly; hence, its
use is limited to small fragments of tissue.
b. Picrates are soluble in water,
hence, tissues should not be washed with running water but rather, transferred
directly from fixative to 70% alcohol.
c. It is not suitable for fixing
kidney structures.
d. It destroys cytoplasmic
structures, e.g. mitochondria
e. It produces RBC hemolysis and
remove demonstrable ferric iron from blood pigments.
f. It reduces or abolishes Feulgen
reaction due to hydrolysis of nucleoproteins.
8. Brasil’s
solution
Advantages:
a. It is better and less “messy”
than Bouin’s solution.
b. It is an excellent fixative for
glycogen.
Overnight tissue fixation by automatic
processing techniques may utilize 3 – 4 changes of Brasil’s fixative at 1 ½ to
2 hours each, succeeded directly by absolute alcohol.
B. Cytological
fixatives
1. Nuclear
fixatives
a. Flemming’s fluid is the most
common Chrome–Osmium Acetic Acid fixative used, recommended for nuclear
preparation of such sections.
Fixation
time: 24 – 48 hours
Advantages:
(1) It
is an excellent fixative for nuclear structures, e.g. chromosomes
(2) It
permanently fixes fat
(3) Relatively
less amount of solution is required for fixation (less than 10 times the volume
of the tissues to be fixed)
Disadvantages:
(1) It
is a poor penetrating agent; hence, is applicable only to small pieces of
tissues.
(2) The
solution deteriorates rapidly and must be prepared immediately before use.
(3) Chromic–Osmic
acid combinations depress the staining power of hematoxylin (especially
Ehrlich’s hematoxylin)
(4) It
has a tendency to form artifact pigments; these may be removed by washing the
fixed tissue in running tap water for 24 hours before dehydration.
(5) It
is very expensive.
b. Carnoy’s fluid – recommended
for fixing chromosomes, lymph glands and urgent biopsies.
Fixation
time: 1 – 3 hours
Advantages:
(1) It
is considered to be the most rapid fixative.
(2) It
fixes and dehydrates at the same time.
(3) It
permits good nuclear staining and differentiation.
(4) It
preserves Nissl granules and cytoplasmic granules well.
(5) It
preserves nucleoproteins and nucleic acid.
(6) It
is an excellent fixative for glycogen since aqueous solutions are avoided.
(7) It
is very suitable for small tissue fragments such as curettage and biopsy
materials.
(8) Following
fixation, tissues may be transferred directly to absolute alcohol, thereby
shortening processing time.
Disadvantages:
(1) It
produces RBC hemolysis.
(2) It
causes considerable tissue shrinkage.
(3) It
is suitable for small pieces of tissues due to slow penetration.
(4) It
tends to harden tissues excessively and distorts tissue morphology.
(5) It
dissolves fat, lipids and myelin.
(6) It
leads to polarization unless very cold temperature (–70oC) are used.
c. Bouin’s fluid (see discussions
above)
d. Newcomer’s fluid
Fixation
time: 12 – 18 hours at 3oC
Advantages:
(1) It
is recommended for fixing mucopolysaccharides and nuclear proteins.
(2) It
produces better reaction in Feulgen stain than Carnoy’s fluid.
(3) It
acts both as nuclear and histochemical fixation.
2. Cytoplasmic
fixatives
a. Fleming’s fluid
without Acetic acid
– made up only of chromic and osmic acid, recommend for cytoplasmic structures
particularly the mitochondria. The removal of acetic acid from the formula
serves to improve the cytoplasmic detail of the cell.
Fixation
time: 24 – 48 hours
Advantages and disadvantages is the same
as Fleming’s solution.
b. Helly’s fluid
Fixation
time: 12 – 24 hours
Advantages:
(1) It
is an excellent microanatomic fixative for pituitary gland, bone marrow and
blood containing organs such as spleen and liver.
(2) It
penetrates and fixes tissues well.
(3) Nuclear
fixation and staining is better than Zenker.
(4) It
preserves cytoplasmic granules.
Disadvantages:
The disadvantages of Helly’s solution
are similar to Zenker’s except that brown pigments are produced if tissues
(especially blood containing organs) are allowed to stay in the fixative for
more than 24 hours due to RBC lysis. This may be removed by immersing the tissue
in saturated alcoholic Picric acid or Sodium hydroxide.
c. Formalin with
“post–chroming”
d. Regaud’s fluid
(Moller’s fluid)
Fixation
time: 12 – 48 hours
Advantages:
(1) It
penetrates tissues well.
(2) It
hardens tissues better and more rapidly than Orth’s fluid.
(3) It
is recommended for demonstration of chromatin, mitochondria, mitotic figures,
Golgi bodies, RBC and colloid– containing tissues.
Disadvantages:
(1) It
deteriorates and darkens on standing due to acidity; hence, the solution must
always be freshly prepared.
(2) Penetration
is slow, hence, tissues should not be thicker than 2 – 3 mm.
(3) Chromate–fixed
tissues tend to produce precipitates of sub–oxide, hence should be thoroughly
washed in running water prior to dehydration.
(4) Prolonged
fixation blackens tissue pigments, e.g., melanin; this may be removed by
washing the tissue in running tap water prior to dehydration.
(5) Glycogen
penetration is poor; it is therefore, generally contraindicated for
carbohydrates.
(6) Nuclear
staining is poor.
(7) It
does not preserve fats.
(8) It
preserves hemosiderin less than buffered formalin.
(9) Intensity
of PAS reaction is reduced.
e. Orth’s fluid
Fixation
time: 36 – 72 hours
Advantages:
(1) It
is recommended for study of early degenerative process and tissue necrosis.
(2) It
demonstrates rickettsia and other bacteria
(3) It
preserves myelin better than buffered formalin.
Disadvantages:
Same as Regaud’s
fluid
C. Histochemical
fixative
1. Formol saline
10%
This is a simple microanatomical
fixative made up of Standard Formaldehyde diluted to 10% with sodium chloride;
it is recommended for fixation of Central Nervous Tissues and general
post–mortem tissues for histochemical examinations.
Fixation
time: 24 hours at 35oC;
48 hours at 20 –
25oC
2. Absolute Ethyl
alcohol
3. Acetone – used at ice
cold temperature ranging from –5oC to 4oC
Advantages:
a. It is recommended for the study
of water diffusible enzymes especially phosphatases and lipases.
b. It is used in fixing brain
tissues for diagnosis of rabies.
c. It is used as a solvent for
certain metallic salts to be used in freeze substitution techniques for tissue
blocks.
Disadvantages:
a. It produces inevitable shrinkage
and distortion.
b. It dissolves fat.
c. It preserves glycogen poorly.
d. It evaporates rapidly.
4. Newcomer’s fluid
Fixation
time: 12 – 18 hours
at 3oC
Advantages:
a. It is recommended for fixing
mucopolysaccharides and nuclear proteins
b. It produces better reaction in
Feulgen stain than Carnoy’s fluid
c. It acts both as a nuclear and
histochemical fixative
Related
fixation term:
1. Secondary
fixation
– is the process of placing an already fixed tissue in a second fixative in
order to:
a. To improve the demonstration of
particular substance
b. To make special staining
techniques possible (with secondary fixative reacting as mordant).
c. To ensure further and complete
hardening and preservation of tissues.
This maybe done before dehydration and
on deparaffinized sections before staining, usually with 10% formalin or 10%
formol saline as a primary fixative.
2. Post–chromatization – secondary
fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5
– 3% potassium dichromate for 24 hours, to act as a mordant for better staining
effects and to aid in cytologic preservation of tissues.
3. Washing out – is the
process of removing excess fixative from the tissue after fixation in order to
improve staining and remove artifacts from the tissues. Several solutions may
be used.
a. Tap water – is used to
remove:
(1) Excess
chromates from tissues fixed in Helly’s, Zenker’s and Fleming’s solution.
(2) Excess
formalin
(3) Excess
osmic acid
b. 50 – 70% alcohol – is used to
washout excess amount of picric acid (Bouin’s solution)
c. Alcoholic iodine – is used to
remove excessive mercuric fixative.
Factors
that affect fixation of tissues:
A. Retarded
by
1. Size and
thickness of the tissue specimen – larger tissues require more fixatives
and longer fixation time.
2. Presence of
mucus
– prevents complete penetration of fixative; hence, tissues that contain mucus
and fixed slowly and poorly. Excess mucus may be washed away with normal saline
solution.
3. Presence of fat – fatty tissues
should be cut in thin sections and fixed longer.
4. Presence of
blood
– tissues containing large amount of blood (e.g. blood vessels and spleen)
should be flushed out with saline by arterial cannulization before fixing.
5. Cold
temperatures
– inactivates enzymes
B. Enhanced
by
1. Size and
thickness of tissues
– smaller and thinner tissues require less fixative and shorter fixation time.
2. Agitation – fixation is
accelerated when automatic or mechanical tissue processing is used.
3. Moderate heat
(37 – 56oC) accelerates fixation but hastens autolytic changes
and enzyme destruction.
PRINCIPLES AND
PRECAUTIONS IN HANDLING AND FIXATION OF SPECIMENS IN GENERAL
1. Autopsy materials should be fixed
as soon after death as possible to prevent decomposition and autolysis due to
deprived oxygen and metabolism. If this is not possible, the body should be
placed in a mortuary refrigerator (kept at temperature of 4oC) or
undergo arterial embalming for better tissue preservation.
2. Surgical specimens should be
fixed as soon as possible after removal or refrigerated if fixation is to be
delayed, to prevent drying of surface layers and ultimate tissue distortion.
If placed in NSS during the operation,
autolysis may occur before fixation is carried on.
3. All tissue specimens must be
properly labeled and identified.
4. If tissues are refrigerated, slow
freezing of unfixed tissues near 0oC must be avoided since this may promote
formation of ice crystal artifacts. Repeated freezing and thawing, on the other
hand, will destroy cellular organelles, release enzymes and diffuse soluble
components of the cell.
5. Tissue slices should be taken at
right angles to the surface of the organ and should be sufficiently deep to
show the normal anatomic components.
6. Tissues should not be more than 5
mm thick except in lung edema (in which case tissue slices may be 1 – 2 cm thick),
with minimum squeezing and handling.
Thin sections allow complete penetration
by fixative in a short time.
7. Purulent materials, exudates or
transudates should be marked and kept for possible cultures, smears and other
bacteriologic examinations.
8. The amount of fixative must be
adequate, approximately 20 – 50 times the volume of the tissue specimen except
in osmium tetroxide which is very expensive requiring only 5 – 10 times that of
tissue volume for fixation.
9. Contamination of fixed tissue
with precipitate (e.g. Osmium tetroxide), should be avoided.
10. In
most instances, fixed tissues must be washed thoroughly to remove salts and/or
pigments before staining.
11. Low
temperature retards fixation but prevents autolysis, therefore tissues should
be fixed at a temperature near the freezing point of the fixative.
12. The
required period for fixation should not be exceeded since this may cause
excessive hardening or brittleness of tissues.
13. There
must always be an adequate supply of fixatives at all times.
14. Drying
should be avoided to prevent shrinkage and distortion of tissue with loss of
cellular detail.
Small tissue biopsies may be placed in a
petri dish with moistened filter paper to prevent drying.
15. Solid
organs should be injected with, as well as immersed in, enough fixatives to
ensure complete penetration and fixation.
16. Hollow
organs (e.g. stomach, intestines) should be packed with cotton soaked in
fixative or completely opened before being immersed in adequate fixing
solution.
17. Air–filled
lungs may float on fixative. To avoid this, the organ may be covered with
several layers of gauze to maintain it under surface.
18. Human
brains should undergo intravascular perfusion (washing out of blood with
Ringer’s lactate). This may, however, lead to artifact formation with loss of
blood content. They may be suspended by a cord tied under the Circle of Willis
to prevent flattening.
19. Dense
tissues are poorly penetrated, hence require long fixation.
20. Eyes
should not be dissected before they are fixed since this may lead to immediate
tissue collapse and wrinkling due to escape of vitreous humor. They are not,
however, easily penetrated due to tough sclera. Formol – alcohol must be
injected before immersing the organ in the fixative.
21. Frozen sections may lead to formation of ice
crystals artifacts.
21. When
fixing muscles, to avoid rigor contraction and staining of artifacts, the fresh
biopsy material may be stretched for 30 minutes by sutures on each end, and
left in a moist filter paper placed in a petri dish or suspended in fixative.
22. Water
should not be used for glycogen–containing tissues because glycogen is soluble
in water.
23. To
assure rapid access of the fixative to all parts of the tissue, the tissue may
be minced, that is, small pieces of specimen may be divided into small
fragments (½ to 1 x 1 mm) and transferred to the vial of a fixative by means of
a toothpick.
24. Hard
tissues (e.g. cervix, uterine, fibroids, hyperkeratotic skin, fingernails,
etc.) may be washed out in running water overnight and immersed in 4% aqueous phenol
solution for 1 – 3 days (Lendrum method). This will soften the tissue and allow
easier sectioning without producing any marked distortion of the cell
structure.
Proper
tissue processing should start with proper fixation and preservation since a
badly preserved tissue will later on yield a badly processed specimen and may
prove to be unsuitable for study. The choice of fixative and mode of processing
must therefore vary depending upon the following factors:
1. Need for immediate examination.
2. Tissue structure or component to
be studied.
3. Type of tissue to be processed.
4. Staining technique to be applied.
5. Type of specimen to be made, whether
serial or individual.
Good
cutting and staining of section is greatly dependent upon proper fixation.
Errors in the choice and use of fixative have to a very large extent adversely
affected and caused ultimate failure in the processing of tissues under
investigation.
Some
of the difficulties encountered because of improper fixation are:
1. Failure to
arrest early autolysis of cells
Cause:
Failure to fix immediately (the tissues
was probably allowed to dry before fixing); Insufficient fixative
2. Removal of
substances soluble in fixing agent
Cause:
Wrong choice of fixative
3. Presence of
artifact pigments on tissue sections
Cause:
Incomplete fixation
4. Tissues are soft
and feather–like in consistency
Cause:
Incomplete fixation
5. Loss or
inactivation of enzymes needed for study
Cause:
Wrong choice of fixative
6. Shrinkage and
swelling of cells and tissue structure
Cause:
Overfixation
7. Tissue blocks
are brittle and hard
Cause:
Prolonged fixation
An incompletely fixed tissue may lead to
improper and incomplete clearing and impregnation and may later prove to be a
hindrance to normal sectioning and staining of specimen.
HOPE:
Herpes–glutamic acid buffer mediated Organic Solvent Protection Effect
The
discovery of the so-called HOPE method allows tissue samples to be treated such
that they do not only meet the requirements of clinical histology, but can still
be characterized later on by modern methods of proteomics, a technique
analyzing all proteins at once. This is successful, since the structure of the
tissue is "fixed" in a way that the protein molecules remain
accessible for systematic analysis. This technique therefore meets current
requirements in terms of a more personalized medicine and thus opens up new
opportunities for researching diseases and their therapies.
HOPE
stands for "Hepes-glutamic acid buffer mediated Organic solvent Protection
Effect" and is a method for preserving tissue samples for later analysis.
A
look at a tissue sample through the microscope tells researchers and
pathologists a whole story about a patient's health status. In order to
preserve the tissue, samples are taken and usually fixed with formalin, before
they are embedded in wax-like paraffin and cut into razor-thin slices. These
are then stained and allow the experienced eye to discern tissue structures and
make diagnoses and prognoses.
One
disadvantage of this type of sample preparation is that formalin cross-links
the protein molecules that are present in the cell. This makes them difficult
to analyze. In order to carry out analyses of this type, snap-frozen samples
are used.
Snap-freeze is a term often
used in scientific papers to describe a process by which a sample is very
quickly lowered to temperatures below –70°C. This is often accomplished by
submerging a sample in liquid nitrogen. This prevents water from crystallizing
when it forms ice, and so better preserves the structure of the sample (e.g.
RNA, protein, or live cells)
