FIXATION

Histotechnology

• The art and science performed by the histot

echnologist to produce a tissue section of go
od quality that will enable the pathologist to
diagnose the presence or absence of disease.
 Fixation

• The first and most critical step in histotechnolo

gy.

• It involves preservation of fresh tissue.

• Prevents degeneration, decomposition, putrefa

ction, and distortion of tissues after removal fro
m the body.
• Fixatives increases the optical differentiation of
cell and tissue components

• Fixatives act as mordants or accentuators to pro

mote and hasten staining or they may inhibit ce
rtain dyes in favor of another

• Fixatives reduces risk of infections during handli

ng and actual processing of tissues.
 Fixation

– The quality of the section on the slide is o

nly as good as the quality of the fixed tissu
e specimen

– If not adequate, other process following fi

xation will not be adequate.
 Fixation
• Primary goal
– To preserve the shape, structure, intercell
ular relationship and chemical integrity an
d constituent of the cell in as life-like man
ner as possible.
• Leaving tissue in air for prolonged period
of time causes drying out and distortion.

• Leaving tissue in water (hypotonic sol’n)

– causes cell to swell

• Leaving tissue in water with high salt (hyp

ertonic) – causes cell to shrink
• Secondary goal:
– To harden and protect the tissue from the
trauma of further handling and make it ea
sier to cut during gross examination.
 Mechanism involved in fixation

• Additive fixation
• Non-additive fixation
 Additive fixation

• The chemical constituent of the fixative is ta

ken in and becomes part of the tissue by for
ming cross-links or molecular complexes and
giving stability to the protein.

– Ex: formalin, mercury and osmium tetroxi

de
 Non-additive fixation

• The fixing agent is not incorporated into the

tissue, but alters the tissue composition and
stabilizes the tissue by removing the bound
water attached to H-bonds of certain group
within the protein molecule

– Alcoholic fixatives
 Factors involved in Fixation:

1. Hydrogen Ion concentration

2. Temperature
3. Thickness of section
4. Osmolality
5. Concentration
6. Duration of fixation
1.)Hydrogen Ion Concentration

• Satisfactory fixation occurs between pH 6 an

d 8.

• Outside the range, changes may occur that a

re detrimental to ultrastructural preservatio
n of the tissue
 2.)Temperature
• Traditionally carried at room temperature(2
2-25oC).

• Many labs use tissue processors that work at

40oC.

• For electron microscopy and some histoche

mistry – 0-4oC
• Nucleic Acids – do not react with fixative to
any extent at room temperature – should be
fixed at higher temperatures.

• For rapid fixation of very urgent biopsy spec

imen – heated to 60oC
– Higher risk of tissue distortion
• Tissues with tuberculosis - formalin at 100oC
is used. (microwave fixation)
3.)Thickness of section
 4.)Osmolality

• Hypertonic fixatives – cell shrinkage

• Isotonic (340mOsm) and hypotonic fixatives –

cause cell swelling and poor fixation

• slightly hypertonic(400-450mOsm) – best to u

se
 5.)Concentration

• Formaldehyde – 10% solution

• Glutaraldehyde – 3% solution
 6.)Duration of Fixation
• Usually 24 hours

• Buffered formalin – 2-6 hours (but may remain for da

ys)

• Prolonged fixation may cause shrinkage and hardeni

ng of the tissue and may severely inhibit enzyme acti
vity and immunologic reactions. (washing in running
water can restore the activity of some enzymes)
• For electron microscopy – fixed for 3 hours a
nd then placed in a buffer
1.)Speed= placed immediately in
PRACTICAL CONSIDERATIONS OF FIXATIONS
formalin after body removal
2.)Penetration= 1mm/hr.
3.)Volume= 20x or 10-25x
4.)Duration=24 hrs.
5.)Size= 2cm2 no more than 4mm
Practical Considerations of Fixation

• Speed – the specimen should be fixed imme

diately to prevent autolysis and putrefaction

• Penetration – Formalin diffuses into the tiss

ue at the rate of approximately 1mm per ho
ur and slows down as it goes deeper into the
tissue
• Volume – Traditionally 10-25 times the volume of t
issue to be fixed; 20 times the tissue – maximum ef
fectiveness.

• Duration of fixation – Fibrous organs such as uteru

s or intestinal track take longer than small or loosel
y textured tissues such as biopsies or scrapings
– Fixation time – speed up using heat, vacuum, ag
itation or microwave
 Good Fixative

• Must be cheap
• Must be stable
• Must be safe to handle
• Must kill the cell quickly – produce minimum
distortion of cell constituents
 Good Fixative

• Must inhibit bacterial decomposition and autol

ysis

• Must produce minimum shrinkage of tissue

• Must permit rapid and even penetration of tiss

ues

• Must harden tissues – cutting of section easier

 Good Fixative

• Must make cellular components insoluble to

hypotonic solutions and render insensitive t
o subsequent processing

• Must permit the subsequent application of

many staining procedures
 Good Fixative

• However, no single fixative has yet known to

possess all the qualities of an ideal solution
FACTORS THAT AFFECTS FIXATION
• Size and Thickness
• Mucus
• Fat
• Blood
• Cold Temperature
 =TYPES OF FIXATIVES=

• I.)According to “COMP
OSITION”

• II.)According to “ACTI
ON”
A.)Simple Fixatives
Aldehyde
I.)According to
-Formaldehyde “COMPOSITION”
-Glutaraldehyde

Metallic Fixative
-Mercuric fixatives -Lead fixatives
-Chromate fixatives

B.)Compound Fixatives
 II.)According to “ACTION”
A.)Microanatomical fixatives- permits general
microscope study of tissue.

Ex.
 10% Formol Saline
 Zenker’s solution
 10% Neutral buffered formalin
 Helly’s Solution(Zenker-formol)
 Heidenhain’s Susa
 Bouin’s solution
 Formol Sublimate
 Brasil’s
B.)Cytological fixatives-preserves specific parts of the
cell

I.)Nuclear fixatives
-Flemmings fluid -Newcomer’s fluid
-Carnoy’s fluid -Heidenhein’s
-Bouin’s fluid

REMEMBER B-E-N-C-H!!!!
Usually contains GLACIAL ACETIC ACID!!!
pH is 4.6 or less
Heated formalin at: 45’C =RNA
65’C=DNA
 II.Cytoplasmic fixatives
-Flemming’s without acetic acid -Regaud’s Muller
-Formalin with “post-chroming” -Orth’s fluid
-Helly’s fluid
REMEMBER F-F-O-R-H !!!!
NEVER contains glacial acetic !!!!
pH should be greater than 4.6
III.Histochemical fixatives- are
those that preserve the chemical
constituents of cells and tissues.
-10% formol saline -Newcomer’s fluid
-Absolute Ethanol -formalin with
calcium(Baker’s)
-Acetone
ALDEHYDE FIXATIVES
1.) Formaldehyde(formalin)

-mixed with water or other compound with dilution of 1:10 or 1:20

-for staining of chromaffin cells in pheochromocytoma

2.) 10%Formol-Saline

-40% formalin diluted with 10% NaCl

-recommended for CNS, and histochemical

3.) 10% Neutral Buffered Formalin/Phosphate-buffered formalin

-buffered to pH 7 with phosphate

-post-mortem,surgical, Iron pigments, Elastic Fibers

4.)Formal Corrosive
-contains HgCl, but does NOT need to be “washed out”.
-Lipids, neutral fats, phospholipids
-silver reticulum methods
5.)Alcoholic Formalin(GENDRE’S FIXATIVE)
-95% Ethyl Alcohol, Picric Acid, Acetic Acid, formalin
-sputum,glycogen REMEMBER !!!!!
6.)Glutaraldehyde
-when buffered with osmic acid is utilized in ELECTRON
MICROSCOPY
-best in CNS, made up of 2 formalin residues linked to 3 carbon
chains.
1.)Formaldehyde(10% formalin)

• Most widely used fixative is 10% formalin.

• A gas produced by the oxidation of methyl al

cohol, and is soluble in water to the extent o
f 37-40% weight(gas) in volume.
• Pure stock solution of 40% formalin is unsatisfactory
for routine fixation – overharden the outer layer of t
he tissue and affect staining adversely.

• Formaldehyde is commonly used as a 4% solution to

give a 10% formalin solution.

• Therefore, a 1:10 or 1:20 dilution is made to make a

10% or 5% solution.
• Fixation time: 24 hours

• Buffered to pH 7 with phosphate buffer

• A “tolerant” fixative, used for mailing spe

cimen because specimen can be left in form
alin indefinitely
 Disadvantages:

• Fumes can cause sinusitis, allergic rhinitis, excessive l

acrimation, allergic dermatitis

• It may produce considerable shrinkage of tissues

• A soft fixative and does not harden some cytoplasmi

c structures adequately enough for paraffin embeddi
ng
• If unbuffered: forms abundant brown pigment granul
es on blood-containing tissues , ex. Spleen, due to bl
ackening of hemoglobin

• Prolonged fixation: Bleaching of the specimen and lo

ss of natural color, dispersal of fat into the fluid and d
issolution or loss of glycogen, biurate of sodium cryst
als and uric acid
 Precautions
• Prolonged storage of formaldehyde especiall
y at very low temperature, may induce preci
pitation of white paraformaldehyde deposit
s and produce turbidity but does not impair
the fixing property of the sol’n

• – precipitates may be removed by filtration

or by addition of 10% methanol
• Concentrated solutions of formaldehyde must
never be neutralized since this might precipitat
e violent explosions

• Room should be properly exhausted – can caus

e injury to eyes and nose.
• Bleaching of tissues may be prevented by ch
anging the fluid fixative every three months.

• Natural tissue colors may be restored by im

mersing tissues in 70% alcohol after fixation
 2.)10% Formol-saline
• Made up of saturated formaldehyde diluted
to 10% with sodium chloride.

• Recommended for fixation of nervous tissue

and general post-mortem tissue for histoche
mical examination.
 Disadvantages:

• Similar to formaldehyde with additions:

• Slow fixative – requires 24hours or longer pe
riod of fixation.

• Tissues tend to shrink during alcohol dehydr

ation.
3.) 10% neutral buffered formalin or
phosphate buffered formalin(pH 7)
• Recommended for preservation and storage
of surgical, post-mortem and research speci
men.

• Fixation time: 4-24 hours

• Advantage:
– Best fixative for tissues containing iron pig
ments and for elastic fibers.
–
• Disadvantage
– It is longer to prepare – time consuming
4.)Formal-Corrosive(formal sublimate)

• Formal-mercuric solution is recommended for r

outine post-mortem tissues

• Formula Sat.Aq. Mercuric Chloride 90mL

Formaldehyde 40% 10 mL

• Fixation time: 3-24 hours

• Advantages:
– Penetrates small pieces of tissues rapidly

– Excellent for many staining procedures inc

luding silver reticulum method
• Disadvantages:

– It forms mercuric chloride deposits

– Slow to penetrate with more than 1 cm in

thickness of tissue section
 5.)Alcoholic Formalin(Gendre’s
fixative)
• Post-fixation with phenol-formalin for 6 hours or more ca
n enhance immunoperoxidase studies on the tissues, and
some cases, electron microscopy.

• Formula:
95% Ethyl Alcohol saturated with picric acid 80mL
Strong formaldehyde soln 15mL
Glacial acetic acid 5 mL
• Advantages:
– Fixation time is reduced to half

– It fixes and Dehydrates at the same time

– Used to fix Sputum!!!

• Disadvantages:
– Causes partial lysis of RBC

– Does not give as good morphological struct

ure as glutaraldehyde.
 6.)Glutaraldehyde

• Made up of two formaldehyde residues, link

ed by three carbon dioxide chains.

• A 2.5% solution is used for small tissue frag

ments and needle biopsies fixed in 2-4 hours.
 Advantages:

• Has more stable effect on tissue – especially

of CNS

• It preserves plasma protein better

• It is less irritating to the nose and does not c

ause dermatitis
 Disadvantages

• More expensive

• Less stable in the environment

• Tends to make tissue more brittle

METALLIC FIXATIVES
 A.)Mercuric Chloride
-most common metallic fixative

-good for cytoplasmic staining and trichome staining !!!!!

a.)Zenker’s fluid- Hgcl, Glacial acetic acid, K2Cr

-liver,spleen, connective tissue

-Immunoglobulins, non-immunohistochem detection of

RHABDOMYSOSARCOMA

b.)Zenker-Formol- HgCl, formaldehyde,K2Cr,

-pituitary gland, REO

 c.)Heidenhain’s Susa- HgCl, formaldehyde,TCA,
Acetic Acid, NaCl

-recommended for TUMOR & SKIN BIOPSIES.

-doesn’t give ppt.

d.)B-5 fixative- HgCl, Na acetate

-used for BONE MARROW, IMMUNOGLOBULINS, RBC’S

 Mercuric chloride
• Most common metallic fixative, frequently u
sed in saturated aqueous solutions of 5-7%.

• Widely used as secondary fixative

• Penetrates poorly and produces shrinkage of

tissues
• Tissues fixed with mixtures containing mercuric chl
oride(except heidenhain’s susa) contain black pre
cipitates of mercury .

• Deposits are removed from deparaffinized sections

before staining by treating the section with 0.5% io
dine sol’n in 70% ethanol for 5-10minutes.
• Sections are rinsed in water, decolorized for
5 minutes in 5% sodium thiosulfate and was
hed in running water.
 Advantages:
• Trichrome staining is excellent

• Routine fixative of choice for preservation of cell d

etail in tissue photography

• Permits brilliant metachromatic staining of cells

• Recommended for renal tissue, fibrin, CT, and mus

cles
 Disadvantages:
• Penetration beyond the first 2-3 mm is slow;

• If left in fixative for more than 1-2 days, the t

issue becomes unduly hard and brittle.

• Extremely corrosive to metals

 Precautions:

• Black deposits may be removed by adding s

aturated iodine solution in 96% alcohol

• The use of metallic forceps and of metal ca

ps to cover the bottles containing the fixati
ve should be avoided
 1.)Zenker’s fluid
• Made up of mercuric chloride stock solution to
which glacial acetic acid has been added just b
efore its use to prevent turbidity and formatio
n of a dark precipitate.

• It is recommended for fixing small pieces of liv

er, spleen, CT fibers and nuclei
• Formula:
Mercuric chloride stock sol’n 95mL
Glacial acetic acid 5mL

• Fixation time: 12-24 hours

 Advantages:

• Stock solutions keep well without disintegrat

ion

• It is recommended for trichrome staining

 Disadvantages:

• Prolonged fixation (more than 24hours) will make t

issues brittle and hard.

• It causes lysis of RBCs and removes iron from hemo

siderin

• Tissue must be washed in running water for several

hours(or overnight) before processing – insufficient
washing may inhibit or interfere with good cellular
staining.
 Practical considerations

• Mercuric deposits may be removed by imme

rsing tissues in alcoholic iodine solution prio
r to staining, through a process known as de
-zenkerization.
 “Dezenkerization”

• Bring slides to water

• Immerse in lugol’s iodine (5 minutes)
• Wash in running water (5min)
• Immerse in 5% sodium thiosulfate (5mins)
• Wash in running water (5mins)
• Proceed with required water soluble stain
2.)Zenker-formol (Helly’s solution)
• Formula:
K2Cr
Distilled water
Strong formaldehyde
Mercuric Chloride stock solution
Mercuric Chloride
Na sulfate
• Fixation time: 12-24 hours
Advantages:
 Disadvantage

• Brown pigments are produced if tissues are

allowed to stay in the fixative for more than
24hours due to RBC lysis – may be removed
y immersing the tissue in
saturated alcoholic picric acid or
sodium hydroxide solution
 3.)Heidenhain’s Susa solution

• Recommended mainly for SKIN TUMOR biop

sies

• Fixation time: 3-12 hours

• Formula:
Mercuric chloride 45 g
NaCl 5g
Trichloroacetic acid 20 g
Glacial acetic acid 40mL
40% formaldehyde 200mL
Distilled water 800mL
 4.) B-5 fixative
• Commonly used for Bone marrow biopsies

• Formula: Dist water 90mL

Mercuric chloride 6g
Sodium acetate 1.25 g

* Just prior to use, add 1mL of 40% formaldehy

de for 10mL of B-5
• Advantage:
– Rapid fixation can be achieved in 1 ½ to 2
hours

• Disadvantage:
– Over-fixation hardens the tissue and make
s cutting difficult
a.)Chromic Acid
CHROMATE
b.)Potassium Dichromate Fixative
c.)Regaud’s /Muller’s Fluid

-K2Cr, Strong Formaldehyde

-mitotic figure, mitochondria

d.)Orth’s fluid

-K dichromate, NaSO4, strong formaldehyde, recommended for

early degenerative process, and tissue necrosis, RICKETTSIA,
CHROMAFFIN GRANULES, Myelin
 Lead Fixatives

-recommended for ACID

MUCOPOLYSACCHARIDES,
Connective tissue MUCIN
-Affinity for basic dyes and Best for GLYCOGEN demonstration
-It may dissolve LIPIDS, May stain yellow with the tissue, but may
removed with the use of Acid dye or Lithium carbonate
-must NEVER be washed with water before dehydration
A.)Bouin’s Solution
-Picric acid, Glacial acetic acid, strong formaldehyde, for EMBRYOS AND
PITUITARY, no need of washing-out
b.)Brasil’s- 37%Formaldehyde, Picric Acid, Ethanol/Isopropyl Alcohol,
Trichloroacetic acid
GLACIAL ACETIC ACID
 Glacial Acetic acid

• Normally used in conjunction with other fixati

ves to form a compound solution.

• Solidifies at 17oC (glacial acetic acid)

• Fixes and precipitates nucleoproteins

ALCOHOLIC FIXATIVE
• fix and preserves GLYCOGEN, PIGMENTS, AND BLOOD

a.)100% Methyl Alcohol-wet smears, blood smears

b.)Ethyl Alcohol

d.)95% Isopropyl Alcohol-fixing TOUCH PREPARATIONS

e.)Carnoy’s Fluid- Absolute Alcohol, Chloroform, Acetic Acid, used

to fix tissue such as brain for diagnosis of RABIES

f.)Newcomer’s Fluid- Isopropyl Alcohol,Propionic Acid, Petroleum

Ether, Acetone, Dioxane, NUCLEAR & HISTOCHEMICAL

FIXATIVE
OSMIUM TETROXIDE FIXATIVES
• Reduced and forms BLACK ppt. in SUNLIGHT
• BEST!!!! In FATS AND LIPIDS
• can cause Conjunctivitis or blindness; need to
be handled in FUME HOOD
a.) Flemming’s Solution-osmic acid, glacial
acetic acid, Chromic acid
b.)Flemming’s without acetic acid
TRICHLOROACETIC ACID
 Trichloroacetic acid

• May be used as a weak decalcifying agent

ACETONE
 Acetone

• Used at ice cold temperature ranging from -5oC to 4o

C

• Recommended for study of water diffuable enzymes

like phosphatases and lipases.

• Used for fixing brain tissues for diagnosis of rabies.

• END…..

“ The Knowledge of
oneself will preserve that
person from vanity.”
-Miguel de Cervantes