FIXATION OF

TISSUE
BY :
GROUP 1
ARIFAH ( 4183141043)
CICILIA ( 4182141019)
FEBIAN HAGANTA ( 4183141036)
HELEN SINAMBELA (4183342005)
 INTRODUCTION
Appropriate fixation of tissues for histological examination is extreme
ly important. Without attention to this process, the range of tests perf
ormed in a modern histopathology laboratory will be rendered ineffec
tive and practically useless.
• The fixative must have the ability to prevent short- and long-term
destruction of the micro-architecture of the tissue by stopping the
activity of catabolic enzymes and hence autolysis, minimizing the d
iffusion of soluble molecules from their original locations.

• Another important characteristic of a good fixative, which helps maintain tissue a

nd cellular integrity, is the inanimation of infectious agents, which helps maintain
tissue and cellular integrity. It is also important to have good toxicological and fla
mmability profiles that permit the safe use of the fixative .
• The fixative should be readily disposable or recyclable and support long-term tiss
ue storage giving excellent microtomy of paraffin blocks, and should be cost effect
ive
TYPES OF FIXATION
1
Physical methods of fixation
• Heat fixation
• Microwave fixation
• Freeze-drying and freeze sub •
stitution
2 Chemical Fixation
• Coagulant fixatives
• Dehydrant coagulant fixatives
• Non-coagulant cross-linking fixatives
• Formaldehyde fixation
• Glutaraldehyde fixation
Osmium tetroxide fixation
 1 Physical methods of fixation

Heat fixation
The simplest form of fixation is heat. Boiling or poaching a
n egg precipitates the proteins and, on cutting, the yolk a
nd egg white can be identified separately. Each compone
nt is less soluble in water after heat fixation than the same
component of a fresh egg.

Picking up a frozen section on a warm microscope slide both

attaches the section to the slide and partially fixes it by heat a
nd dehydration. Even though adequate morphology could b
e obtained by boiling tissue in normal saline, in histopatholog
y heat is primarily used to accelerate other forms of fixation a
s well as the steps of tissue processing.
1 Physical methods of fixation

Microwave fixation
Microwave heating speeds fixation and can reduce times for fixatio
n of some gross specimens and histological sections from more tha
n 12 hours to less than 20 minutes .Microwaving tissue in formalin r
esults in the production of large amounts of dangerous vapors, so i
n the absence of a hood for fixation, or a microwave processing sys
tem designed to handle these vapors, this may cause safety proble
ms.
Recently, commercial glyoxal-based fixatives which do not form va
pors when heated at 55°C have been introduced as an efficient met
hod of microwave fixation.
1 Physical methods of fixation

Freeze-drying and freeze substitution

Freeze-drying is a useful technique for studying soluble materials and s
mall molecules; tissues are cut into thin sections, immersed in liquid nitr
ogen, and the water is removed in a vacuum chamber at −40°C. The tissu
e can be post-fixed with formaldehyde vapor. In substitution, specimens
are immersed in fixatives at −40°C, such as acetone or alcohol, which slo
wly remove water through dissolution of ice crystals, and the proteins ar
e not denatured; bringing the temperature gradually to 4°C will complete
the fixation process (Pearse 1980).
2. CHEMICAL FIXATION
1
Both organic and non-organic solutions may coagulate
proteins, making them insoluble. Cellular architecture is
maintained primarily by lipoproteins and by fibrous proteins
such as collagen; coagulating such proteins maintains tissue
histomorphology at the light microscopic level.
COAGULANT
FIXATIVES

Unfortunately, because coagulant fixatives result in

cytoplasmic flocculation as well as poor preservation
of mitochondria and secretory granules, such
fixatives are not useful in ultrastructural analysis.
 2. CHEMICAL FIXATION

The most commonly used coagulating fixatives are alcohols (e.g. ethan
ol, methanol) and acetone. coagulant fixation begins at a concentratio
2
n of 50–60% for ethanol but requires a concentration of 80% or more
for methanol. Removal and replacement of free water from tissue by a
ny of these agents has several potential effects on proteins within the
tissue.

DEHYDRANT COAGUL
ANT FIXATIVES
By removing water, the opposite principle weakens hydrophobic bon
ding. Similarly, molecules of water participate in hydrogen bonding i
n hydrophilic areas of proteins; so removal of water destabilizes this
hydrogen bonding. Together, these changes act to disrupt the tertia
ry structure of proteins.
 2. CHEMICAL FIXATION
2

Factors that influence the solubility of macromolecules include:

1. Temperature, pressure, and pH.
2. Ionic strength of the solute.
3. The salting-in constant, which expresses the contribution of th
DEHYDRANT COAGUL e electrostatic interactions.
ANT FIXATIVES
4. The salting-in and salting-out interactions.
5. The type(s) of denaturing reagent(s) (Herskovits et al. 1970; Ho
robin 1982; Papanikolau & Kokkinidis 1997; Bhakuni 1998).
Alcohol denatures protein differently, depending on the choice a
nd concentration of alcohol, the presence of organic and non-or
ganic substances, and the pH and temperature of fixation.
 2. CHEMICAL FIXATION
2

Trichloroacetic acid (Cl3CCOOH) can penetrate hydrophobic do

mains of proteins and the anion produced (–C–COO− ) reacts wit
h charged amine groups. This interaction precipitates proteins an
OTHER TYPES OF COA d extracts nucleic acids. Picric acid or trinitrophenol slightly dissol
GULANT FIXATIVE
ves in water to form a weak acid solution (pH 2.0).

In reactions, it forms salts with basic groups of proteins, causing t

he proteins to coagulate. If the solution is neutralized, precipitate
d protein may redissolve. Picric acid fixation produces brighter st
aining, but the low pH solutions of picric acid may cause hydroly
sis and loss of nucleic acids
 2. CHEMICAL FIXATION
3

Several chemicals were selected as fixatives secondary to their pot

ential actions of forming cross-links within and between proteins a
nd nucleic acids as well as between nucleic acids and proteins. Cros
NON-COAGULANT
slinking may not be a major mechanism at current short times of fix
CROSS-LINKING
ation, and therefore ‘covalent additive fixatives’ may be a better na
FIXATIVES me for this group.
Aldehyde groups are chemically and biologically reactive and are re
sponsible for many histochemical reactions, e.g. free aldehyde grou
ps may be responsible for argentaffin reactions (Papanikolau & Kok
kinidis 1997).
Formaldehyde Fixation

 Formaldehyde in its 10% neutral buffered form (NB

F) is the most common fixative used in diagnostic p
athology.
 37–40% formaldehyde in this aqueous solution is k
nown as ‘formalin’.
 The usual ‘10% formalin’ used in fixation of tissues i
s a 10% solution of formalin, contains about 4% wei
ght to volume of formaldehyde.

 Fraenkel-Conrat and his colleagues  identified

most of the reactions of formaldehyde with amino
acids and proteins.
 Kok & Boon 2003; Leong 2005  Formaldehyde
also reacts with nuclear proteins and nucleic acids.
Schematic View of Formaldehyde
Click to enter title text
Fixation Figure 1.

Researchgate.net
Reversibility of formaldehyde
macromolecular reactions
 The reactive groups may combine with hydrogen groups
or with each other, forming methylene bridges. If the for
malin is washed away, reactive groups may rapidly return
to their original states, but any bridging that has already
occurred may remain.
 Crosslinking is a relatively slow process, so, in the rapid fix
ation used in diagnostic pathology, most ‘fixation’ with for
maldehyde prior to tissue processing stops with the form
ation of reactive hydroxymethyl groups.

 The principal type of cross-link in short-term fixation is

thought to be between the hydroxymethyl group on a l
ysine side chain and arginine (through secondary amin
o groups), asparagine, glutamine (through secondary a
mide groups), or tyrosine (through hydroxyl group) (To
me et al. 1990).
 Formaldehyde primarily preserves peptide protein bond
s and the general structure of cellular organelles.
 Click to enterCrosslinking
Formaldehyde title text

FIGURE 2. Graphic depicting the main aspects of form FIGURE 3. Chemical reactions occurring during formaldehyde cro
aldehyde reactivity in cells. Jbc.org sslinking of biomolecules. Jbc.org.
 Click to enter title
Glutaraldehyde text
fixation

Figure 4.
Glutaraldehyde is a bifunctional aldeh
yde that probably combines with the s
ame reactive groups as does formald
ehyde.

In aqueous solutions glutaraldehyde p

olymerizes, forming cyclic and oligom
eric compounds (Hopwood 1985), an
d it is also oxidized to glutaric acid, an
d To aid in stability, it requires storage
at 4°C and at a pH of around 5 .

Like formaldehyde, reactions with lysi

ne are the most important for forming
cross-links. Glutaraldehyde Fixation.
Glutaraldehyde does not react with ca ScienceDirect.com
rbohydrates or lipids unless they cont
ain free amino groups as are found in
some phospholipids (Hayat 1981).
 Osmium
Click totetroxide fixation
enter title text
Figure 5.
Osmium tetroxide (OsO4), a toxic solid, is solubl
e in water as well as non-polar solvents and can
react with hydrophilic and hydrophobic sites incl
uding the side chains of proteins, potentially cau
sing crosslinking (Hopwood et al. 1990).

Osmium tetroxide is known to int

eract with nucleic acids, specificall
y with the 2,3-glycol moiety in ter
minal ribose groups and the 5,6
double bonds of thymine residues

Nuclei fixed in OsO4 and dehydrated with

alcohol may show prominent clumping of
DNA.
Osmium Tetraoxide.
The best characterized reaction of ScienceDirect.com
osmium is its reaction with unsatura
ted bonds within lipids and phosph
olipids.
 Cross-linking fixatives
Click to enter title text
for electron microscopy

Cell organelles such as cytoplasmic and nuclear me

mbranes, mitochondria, membrane-bound secretory
granules, and smooth and rough endoplasmic reticu
lum need to be preserved carefully for electron micr
oscopy.

The preferred fixatives are a strong crosslinking fixativ

e such as :

 glutaraldehyde,
 a combination of glutaraldehyde formaldehyde, or
 Carson’s modified Millonig’s,

followed by post-fixation in an agent that further stab

ilizes as well as emphasizes membranes such as OsO4.
 Click to enter
MERCURIC title text
CHLORIDE

Historically, mercuric chloride was greatly favor

ed for its qualities of enhancing the staining pr
operties of tissues, particularly for trichrome st
ains.

A further major disadvantage of mercuric

chloride fixation is the inevitable formatio
n of deposits of intensely black precipitat
es of mercuric pigment in the tissues.

Mercury fixatives (Hopwood 1973) are no long

er used routinely except by some laboratories f
or fixing hematopoietic tissues (especially B5).

Special formulations of zinc sulfate in for

maldehyde replacing mercuric chloride in
B5 may give better nuclear detail than for
maldehyde alone and improve tissue pen
etration (Carson 1990).
Figure 6. Mercuric Chloride.
ScienceDirect.com.
 Click to enter title text
Dichromate and chromic acid
fixation

Chromium trioxide dissolves in water to produce an

acidic solution of chromic acid, with a pH of 0.85. C
hromic acid is a powerful oxidizing agent which pro
duces aldehyde from the 1, 2-diglycol residues of p
olysaccharides.

These aldehydes can react in histochemical stains (P

AS and argentaffin/argyrophil)

Fixatives containing chromate at a pH of 3.5–5.0 ma

ke proteins insoluble without coagulation.

Dichromate-containing fixatives have primarily been Figure 7. Comparison of 0-17 M solutions of

used to prepare neuroendocrine tissues for staining, chromic acid and potassium dichromate. Sc :
especially normal adrenal medulla and related tumo Casselman—Cytological Fixation by Chromic
Acid
rs (e.g. phaeochromocytomas)
 Click to enter title text
Fixatives for DNA, RNA, and
protein analysis

Preservation of DNA and RNA and proteins in tissue

s for immunocytochemical analysis using HOPE (HE
PES-glutamic acid buffer mediated Organic Solvent
Protection Effect) fixative and the reversible cross-li
nker dithio-bis[succinimidyl propionate] (DSP)

A novel zinc formation (Z7) containing zinc trifluoro

acetate, zinc chloride and calcium acetate was signif
icantly better than the standard zinc-based fixative
(Z2) and NBF for DNA, RNA and antigen perseverati
Figure 8. Scores from liver, spleen and colon
on. tissue fixed in NBF, Z2, Z4, Z7 and fresh-froze
n tissue (Lykidis., et al. 2007)

Moreover, the fixative is less toxic than formaldehyd

e formulations.
 Click to enter
Metallic title
ions as text supplement
a fixative

Several metallic ions have been used as aids in fixation, including Hg2+ , Pb2+ , Co2+ , C
u2+ , Cd2+ , [UO2] 2+ , [PtCl6] 2+ , and Zn2+ . Mercury, lead, and zinc are used most co
mmonly in current fixatives, e.g. zinc containing formaldehyde is suggested to be a bette
r fixative for immunohistochemistry than formaldehyde alone. This does however depend
upon the pH of the formaldehyde, as well as the zinc formaldehyde (Arnold et al. 1996; E
ltoum et al. 2001a).
.
Compound fixatives
Compound fixatives are useful for specific tissues, e.g.
alcoholic formalin for fixation of some fatty tissues, su
ch as breast, in which preservation of the lipid is not i
mportant. In addition, fixation of gross specimens in al
coholic formalin may aid in identifying lymph nodes e
mbedded in fat.
Factors affecting the quality of fixation
The several factors that will affect the tissue fixatio
n process are as follows:

• Buffers and pH
• Duration of fixation and size of spe
cimens
• Temperature of fixation
• Concentration of fixative
• Osmolality of fixatives and ionic co
mposition
• Additives
Buffers and pH
In fixation, pH is very important. Th
is is especially the case with formal
dehyde, the optimal formalin fixati
on is through buffering at pH 7,2 -
7,4 (ie neutral buffered formalin).
Buffers are used to maintain optimum
pH. The choice of specific buffer depen
ds on the type of fixative and analyte.
Commonly used buffers are phosphate,
cacodylate, bicarbonate, Tris, and acetat
e.
Duration of fixation and size of specimens

• the factors that govern diffusion of a fixative into tissue were investigated by Meda
war (1941).
• he found that the depth (d) reached by a fixative is directly proportional to the squ
are root of duration of fixation (t) and expressed this relation as:

d=k√t

• the constant (k) is the coefficient of diffusability, thus, for most fixatives, the time of
fixation is approximately equal to the square of the distance which the fixative must
penetrate.
• most fixatives, such as NBF, will penetrate tissue to the depth of approximately 1 m
m in one hour; hence for a 10 mm sphere, the fixative will not penetrate to the cen
ter until (5)2 or 25 hours of fixation, it is important to note that the components
of a compound fixative will penetrate the tissue at different rates, so that these
aspects of the fixative will be best manifest in thin specimens.
Temperature of fixation

• the diffusion of molecules increases with rising temperat

ure due to their more rapid movement and vibration;
• the rate of penetration of a tissue by formaldehyde is fa
ster at higher temperatures;
• most chemical reactions occur more rapidly at higher te
mperatures and therefore formaldehyde reacts more rap
idly with proteins.
Concentration of fixative

• effectiveness and solubility primarily determine the a

ppropriate concentration of fixatives.
• concentrations of formalin above 10% tend to cause incr
eased hardening and shrinkage.
• in addition, higher concentrations result in formalin bein
g present in its polymeric form,
• ethanol concentrations below 70% do not remove free
water from tissues efficiently.
Osmolality of fixatives and ionic composition

• the osmolality of the buffer and fixative is important; hy

pertonic and hypotonic solutions lead to shrinkage and
swelling, respectively.
• Similarly, various ions (Na+, K+, Ca2+, Mg2+) can affect
cell shape and structure regardless of the osmotic effect,
the ionic composition of fluids should be as isotonic as
possible to the tissues.
Additives

• The addition of electrolytes and non-electrolytes to fixatives improves the morp

hology of the fixed tissue.
• These additives include calcium chloride, potassium thiocyanate, ammoniu
m sulfate, and potassium dihydrogen phosphate.
• The electrolytes may react either directly with proteins causing denaturation, or
independently with the fixatives and cellular constituents.
• The choice of electrolytes to be added to fixatives used on a tissue processor
may vary.
• Fixatives buffered with electrolytes such as phosphates may cause problems wit
h some processors due to precipitation of the salts.
• The addition of non-electrolyte substances such as sucrose, dextran, and deter
gent has also improve fixation.
Selecting or avoiding specific fixatives

Specific fixatives are unsuitable for most uses and should be avoi
ded. The main problem with fixatives used in histological staining
is the loss by solution/extraction of molecules that are targets of s
pecific histochemical methods. Typically, some molecules are solu
ble in aqueous fixatives (e.g. glycogen), while others are soluble in
organic-based fixatives (e.g. lipids). Some fixatives may chemically
modify targets of histochemical staining and thus affect the qualit
y of special stains (e.g. glutaraldehyde for silver stains); this includ
es modification of staining secondary to changes in pH induced b
y fixation.
Selecting or avoiding specific fixatives
Specific fixatives are unsuitable for most uses and should be avoide
d. The main problem with fixatives used in histological staining is t
he loss by solution/extraction of molecules that are targets of speci
fic histochemical methods. Typically, some molecules are soluble in
aqueous fixatives (e.g. glycogen), while others are soluble in organi
c-based fixatives (e.g. lipids). Some fixatives may chemically modify
targets of histochemical staining and thus affect the quality of speci
al stains (e.g. glutaraldehyde for silver stains); this includes modifica
tion of staining secondary to changes in pH induced by fixation.
USEFUL FORMULAE FOR
FIXATIVE

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1. Neutral Buffered Formalin ( NBF ) 10%

Composition:
 Tap water 900 ml
 Formalin (37% 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

Composition:

 Formaldehyde (37–40%) 10 ml
 Tap water 90 ml
 Sodium phosphate, monobasic 1.86 g
 Sodium hydroxide 0.42 g
 The pH should be 7.2–7.4.
 Better for ultrastructural preservation
3. Formal (10% formalin), Calcium acetate

 Tap water 900 ml

 Formaldehyde (37%) 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 (37%) 100 ml
• Sodium chloride 9 g
5. Formal (10% formalin), zinc, unbuffered

 Tap water 900 ml

 Formaldehyde (37%) 100 ml
 Sodium chloride 4.5 g
 Zinc chloride 1.6 g or zinc sulfate 3.6 g
 Zinc formalin is reported to be an excellent fixative
for immunohistochemistry.
6. Formalin, buffered saline

 Tap water 900 ml

 Formaldehyde (37%) 100 ml
 Sodium chloride 9 g
 Sodium phosphate, dibasic 12 g
7. Formalin, buffered zinc

Neutral buffered formalin 1000 ml

Zinc chloride 1.6 g
 Mercuric fixatives
Problem : Pigments can combine
with mercury.
These pigments are removed fro
m sections by using iodine treatm
ent followed by sodium thiosulfat
e.
1. Zenker’s solution
 Distilled water 250 ml
 Mercuric chloride 12.5 g Potassium dichro
mate 6.3 g Sodium sulfate 2.5 g
 Just before use add 5 ml of glacial acetic a
cid to 95 ml of above solution.
This is a good fixative for bloody (congested)
specimens and trichrome stains.
2. Helly’s solution • 3. scHaudinn’s solution

• Distilled water 250 ml Distilled water 50 ml

• Mercuric chloride 12.5 g Mercuric chloride 3.5 g
• Potassium dichromate 6.3 g Absolute ethanol 25 ml
• Sodium sulfate 2.5 g
Just before use add 5 ml of 37% fo
rmaldehyde to 95 ml of above solu
tion.
It is excellent for bone marrow extram
edullary hematopoiesis and intercalate
d discs.
4. oHlmacHer’s solution
 Absolute ethanol 32 ml
 Chloroform 6 ml
 Glacial acetic acid 2 ml
 Mercuric chloride 8 g
This fixative penetrates rapidly.
5. Carnoy-Lebrun solution
• Absolute ethanol 15 ml
• Chloroform 15 ml
• Glacial acetic acid 15 ml
• Mercuric chloride 8 g
6. B5 fixative
Mercuric chloride 12 g Sodiu
m acetate 2.5 g Distilled wate
r 200 ml
Add 2 ml of formaldehyd
e (37%) to 20 ml of stock
solution just before use.
Frequently used for bone marrow, lymph node
s, spleen, and other hematopoietic tissues.
Dehydrant fixatives
Function: • Components:
to remove free and bound wate Ethanol, absolute Ethanol, 9
r, causing a change to the terti 5%
ary structure of proteins so that
Ethanol, 70–95% Methanol, 1
they precipitate, leaving the nu 00%
cleic acids relatively unchanged.
It is used for immunohistochemistry, enz Acetone, 100%
yme studies, and in the detection of rabi
es.
1. Clarke’s solution
Absolute ethanol 60 ml
Glacial acetic acid 20 ml
This solution produces good gen
eral histological results for H&E
stains.
Advantage of preserving nucleic
acids while lipids are extracted.
A short fixation is recommended
and tissues are transferred to 95
% ethanol following fixation.
2. Carnoy’s fixative
Acetic acid 10 ml
Absolute ethanol 60 ml
Chloroform 30 ml
Carnoy’s fixative is useful for RNA stains, e.
g. methyl green pyronine, and for glycoge
n preservation, in cytology to clear heavily
blood-stained specimens
It shrinks and hardens tissues and hemolyz
es red blood cells.
It may destroy the staining of acidfast baci
lli.
• 3. Methacarn
• Acetic acid 10 ml
• 100% methanol 60 ml
• Chloroform 30 ml
• Causes less hardening and less shrinkage than Carnoy’s, but
with the same pattern of staining.
 Dehydrant cross-linking fixatives
Alcohol-formalin fixation or post-fixation can be advantageous in large specimens
with extensive fat.
A. Alcoholic formalin
1. Ethanol (95%) 895 ml
2. Formaldehyde (37%) 105 ml
B. Alcohol-formalin-acetic acid fixative
Ethanol (95%) 85 ml
Formaldehyde (37%) 10 ml
Glacial acetic acid 5 ml
Methanol may be substituted for ethanol with care; similarly, various mixtures of e
thanol, acetic acid, and formalin may be used.
C. Alcoholic Bouin’s (Gendre’s solution)
Fixation should be between 4 hours and overnight followed by washing in 70% et
hanol, followed by 95% ethanol (several changes). This is the one alcoholic fixative
that improves upon aging
D. Gendre’s solution
95% ethanol saturated with picric acid (5 g per 100 ml) 800 ml
Formaldehyde (37%) 150 ml
Glacial acetic acid 50 ml

To increase the effectiveness of alcoholic Bouin’s, if there is no time for aging, the
following formula has been recommended .
E. Equivalent to aged alcoholic Bouin’s
Picric acid 0.5 g
Formaldehyde (37%) 15 ml
95% ethanol 25 ml
Glacial acetic acid 5 ml
Ethyl acetate 25 ml
Tap water 30 ml
F. Another alcoholic form of Bouin’s solution
Stock Bouin’s solution 75 ml
95% ethanol 25 ml

This solution is excellent for lymph nodes (24 hours)

and for fatty tissue (48 hours).
G. Rossman’s solution
Tap water 10 ml
Formaldehyde (37%) 10 ml
Absolute ethanol 80 ml
Lead nitrate 8 g

Fix for 24 hours at room temperature. This is a good

fixative for connective tissue mucins and umbilical
cord.
For metabolic bone disease
Phosphate buffer
Tap water 1000 ml
NaH2PO4·H2O 1.104 g
NaHPO4 (anhydrous) 4.675 g

Fixative
Phosphate buffer 900 ml
Formaldehyde (37%) 100 ml
Adjust pH to 7.35
Bouin’s decalcifying solution
Saturated aqueous solution of picric acid
(10.5 g per 500 ml) 500 ml
Formaldehyde (37%) 167 ml
Formic acid 33 ml
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.
Fixation for selected individual tissues

Eyes Brain Breast

Lungs Lymphoid tissue Testis

Muscle biopsies Renal biopsies

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