FIXATION
MECHANISMS OF FIXATION
What is tissue processing?
 A tissue taken from the body for
diagnosis must be process in
histopathology laboratory to
produce microscopic slides that
are viewed under the microscope
by the pathologists.
Tissue Processing
A. Fixation
B. Dehydration
C. Clearing
D. Infiltration/Impregnation
E. Embedding
F. Trimming
G. Cutting/Sectioning
H. Staining
I. Mounting
J. Labelling
Fixation
 A surgical specimen fixing in
formalin and ready for grossing
First and most critical step
Aim: to prevent decay and preserve
cells and tissues in a “life-like”
state. Stops enzyme activity, killing
microorganisms and hardening specimen
while maintaining sufficient of the
molecular structure to enable
appropriate staining methods to be
applied
 Inactivation of lysosomal
hydrolytic enzymes – post-mortem
decomposition (autolysis); or by
chemically altering,
stabilizing, and making tissue
components insoluble
 Reducing the risk of infection
by preventing putrefaction after
death (bacterial/fungal
colonization & overgrowth)
Goal: to harden and protect the tissue
from trauma of further handling
 Ideal volume of fixative is 10
to 20 times greater than the
size or volume of the specimen
 Promotes staining
CLASSIFICATION OF FIXING AGENTS &
There are two basic mechanisms
involved in fixation:
 Additive fixation
 Non-additive fixation
Sample
 Physical agent like vacuum, oven
(heat) and agitation increases
movement of molecules and
accelerate fixation
 Used to accelerates training,
decalcification,
immunohistochemistry and
electron microscopy
Advantages:
 Tissue is heated right through
the block in a very short time
(main advantage)
 Non-chemical techniques (less
interference)
 Rapid
 Lesser time for
immunohistochemistry and in-
situ hybridization
Disadvantages:
 Penetrates 10-15mm only
 No significant cross linking
of protein molecules;
subsequent chemical fixation
may be needed
Factors involved in fixation:
 Hydrogen ion concentration
 Temperature
 Thickness of section
 Concentration
 Duration of fixation
Effects of fixatives:
 Harden/soft and friable tissue
 Makes cell resistant to damage
and distortion
 Inhibit bacterial decomposition
 Increase optical differentiation
of cells and tissue components
 Acts as mordant or accentuator
 Reduce risk of infection
Characteristics of a Good Fixative: Cytological Fixatives
 Rapid action and quick Nuclear Cytoplasmic
penetration Flemming’s Flemming’s w/o
 Cheap and economical acetic acid
 Isotonic to the tissue Carnoy’s Helly’s
Bouin’s Formalin w/
 Stable
post chroming
 Should cause minimal loss in the Newcomer’s Regaud’s
physical and chemical properties (Moller’s)
of the tissue
Heidenhain’s Orth’s
 Safe to handle
 Kills quickly Common Fixative Used:
 Hardens tissues for easier
cutting I am Formalin/Aldehyde – Containing
Fixatives:
TYPES OF FIXATIVES  Glutaraldehyde
 10% formaldehyde or 10% formalin
According to composition:
 10% formol saline solution
 Simple
 Kaisserling’s animal cells
 Compound
According to action: Formaldehyde Precautions:
 Micro-anatomical  Formation paraformaldehyde
 Cytological: nuclear &  Well ventilated room
cytoplasmic
 Not neutralized if concentrated
 Histochemical
 Buffered or neutralized by
Simple Fixatives:
adding magnesium carbonate
 Aldehydes
 Changing formalin can prevent
 Formaldehyde bleaching
 glutaraldehyde
Metallic Fixatives: 10% Neutral Buffered
 mercuric chloride Formalin/Phosphate Buffered Formalin
 chromate fixatives  10% Neutral-Buffered Formalin
 Pb Fixatives (1000ml)
Picric Acid o Formaldehyde 100ml
Acetic Acid o Distilled water 900ml
Acetone o Sodium phosphate, monobasic
Alcohol 4g
Osmium Tetroxide/Osmic Acid o Sodium phosphate, dibasic
Heat 6.5g

Types of Fixative 10% Neutral Buffered Formalin

Optimal choice for many reasons
Microanatomical Histochemical  Fast penetration
Fixatives Fixatives  Prevents alterations during
10% Formol Saline Formol Saline 10% processing
10% Neutral Absolute Ethyl
Buffered Formalin Alcohol  Less shrinkage than other
Heidenhain’s Susa Acetone fixatives
Formol Sublimate Newcomer’s fluid  Hardens tissue better
(Formol Corrosive)  Tissue can be stored in formalin
Zenker’s indefinitely
Zenker-Formol  Fixative of choice:
(Helly’s)
immunohistochemistry & molecular
Bouin’s
Brasil’s
tests
 Readily available
Sodium dihydrogen phosphate
 For preservation and storage of
surgical, post-mortem and
research specimens
*best fixative for Fe pigments,
elastic fibers
10% formol saline
 Penetrates and fixes tissues
well, minimum shrinkage &
distortion, does not overharden
tissues
 Slow (>24 h)
Metallic Fixative
A. Mercury Containing
 Most common metallic
fixative
 Routine fixative of choice
for preservation of cell
detail in tissue
photography
 Recommended for renal
tissues, fibrin, CT,
muscles
 Disadvantages: hardens
outer layers only, black
granular deposits formed,
corrosive to metals
Zenker’s Fluid
 Recommended for fixing small
pieces of liver, spleen,
connective tissue fibers, and
nuclei
 Good general fixative for
adequate preservation of all
kinds of tissue and gives
excellent staining result
Zenker Formol (Helly’s Solution)
 An excellent microanatomic
fixative for pituitary gland, BM
& b calllood containing tissue
such as the liver and spleen
 Made up of HgCI2 and
formaldehyde
Heidenhain’s Susa Solution
 Recommended for tumor biopsy
 Made up of MGCI12, glacial
acetic acid and formalin
B-5 Fixative
 Commonly used for BM biopsy
 HgCI12, anhydrous Na acetate
Schaudinn’s fixative
 Alcohol-containing mercury
fixative
 Used for wet smear preparation
and connective tissues
CHROMATE FIXATIVES
Chromic Acid
 Precipitates all CHON and
adequately fixes carbohydrates
Potassium Dichromate (K2Cr2O7)
 Preserves lipid and mitochondria
Regaud’s (Muller’s) Fluid (3%) K2Cr2o7
 Recommended for demonstration of
chromatin, mitochondria, mitotic
figures, golgi bodies, RBC and
colloid containing tissue
Orth’s Fluid (2.5% K2Cr2O7)
 Recommended for study of early
degenerative processes and
tissue necrosis
LEAD CONTAINING
Lillie’s Fixative – used for
preservation of glycogen
mucopolysaccharide, and amyloid
*fixes connective tissue mucin
PICRIC ACID FIXATIVES
A. Picric Acid
 Preserves glycogen well but
causes considerable tissue
shrinkage
 Allows brilliant staining
B. Bouin’s Solution
 Recommended for fixation of
embryos and pituitary
biopsiy
 For embryos, glycogen, not
good for kidneys,
mitochondria, hemolyzes RBC
C. Brasil’s Alcoholic Picroformol
Fixative
 Excellent fixative for
glycogen
*highly explosive when dry
Osmium Tetroxide (Osmic Acid)
 Preserves cytoplasmic structures
well
 Used extensively for
neurological tissues
 Fixes fats
  Inhibits hematoxylin
 Extremely volatile
Flemming’s Solution
 Most common chrome-osmium acetic
acid and fixative
 Excellent fixative for nuclear
structure
 Flemming’s Solution without
Acetic Acid
 Recommended for cytoplasmic
structures
ALCOHOL FIXATIVES
Methyl Alcohol
 Excellent for fixing dry and wet
smears, blood smears and bone
marrow tissues
Ethyl Alcohol
 Used at concentrations of 70-
100%
 Lower concentration causes RBC’s
to be hemolyzed and WBC’s are
inadequately preserved
Gendre’s Fixative
 Sputum
 Made up of alcoholic formalin
OTHER FIXATIVES
Acetone
 It is used in fixing brain
tissues for the diagnosis of
rabies
 Dissolves fat, evaporates
rapidly, preserves glycogen
poorly
Heat Fixation
 Involves thermal coagulation of
tissue proteins
 Usually employed for frozen
tissue sections and preparation
of bacteriologic smear
Carnoy’s Fluid
 Recommended for fixing
chromosomes, lymph glands, and
urgent biopsy
 Made up of ethyl alcohol,
glacial acetic acid and
chloroform
Newcomer’s Fluid
 Recommended for fixing
mucopolysaccharides and nuclear
protein
 Acts both as nuclear and
histochemical fixative
FACTOR AFFECTING FIXATION
Retarded by:
 Size and thickness
 Presence of mucus
 Presence of fats
 Presence of blood
 Cold temperature
Enhanced by:
 Size and thickness
 Agitation
 Moderate heat (37 to 56 degrees
C)
Difficulties caused by improper
fixation:
 Failure to arrest early
autolysis of cells: failure to
fix immediately (the tissue was
probably allowed to dry before
fixing); insufficient fixative
 Removal of substances soluble in
fixing agent: wrong choice of
fixative
 Presence of artifact pigments on
tissue sections: incomplete
washing of fixative
 Tissues are soft and feather-
like in consistency: incomplete
fixation
 Loss or inactivation of enzymes
needed for study: wrong choice
of fixative
 Shrinkage and swelling of cells
and tissue structure:
overfixation
 Tissue blocks are brittle and
hard: prolonged fixation
INCOMPLETE FIXATION
 Results in:
o Separation of tissue
components on the flotation
bath during microtomy
o Poor tissue morphology
o Smudgy nuclei with no
chromatin pattern defined
 o Nuclear bubbling
o Center of tissue more
eosinophilic than periphery

INCOMPLETE FIXATION TROUBLESHOOTING

REMEMBER: the most important pre-

analytical variable
 Amount of time the tissue is not
immersed in the right solution
of formalin (10% neutral
buffered formalin)
 Right volume exposed properly to
formalin by opening, cutting or
bisecting specimens before
immersing in formalin
 Tissue that is not fixed well
does not process well, and
subsequently will not stain well

SECONDARY FIXATION – process of

placing the previously fixed tissue in
a second fixative
Purpose: to improve the demonstration
of a particular substance

POST CHROMATIZATION
 Process wherein a fixed tissue
is placed in an aqueous solution
of 2.5% to 3% potassium
dichromate for 24 hours
 Potassium dichromate acts as
mordant for better staining
effect and preservation of
tissues

WASHING OUT
 Done to remove excess formalin
or fixative from the tissue
after fixation for better
staining effect and to remove
the artifacts from the tissue
using:
o Tap water
o Alcoholic iodine
o 50-70% alcohol