FIXATION CLASSIFICATION OF FIXING AGENTS &
MECHANISMS OF FIXATION
What is tissue processing?
A tissue taken from the body for There are two basic mechanisms
diagnosis must be process in involved in fixation:
histopathology laboratory to Additive fixation
produce microscopic slides that Non-additive fixation
are viewed under the microscope
by the pathologists. Sample
Physical agent like vacuum, oven
Tissue Processing (heat) and agitation increases
A. Fixation movement of molecules and
B. Dehydration accelerate fixation
C. Clearing
Used to accelerates training,
D. Infiltration/Impregnation
decalcification,
E. Embedding
immunohistochemistry and
F. Trimming
electron microscopy
G. Cutting/Sectioning
Advantages:
H. Staining
I. Mounting Tissue is heated right through
J. Labelling the block in a very short time
(main advantage)
Fixation Non-chemical techniques (less
A surgical specimen fixing in interference)
formalin and ready for grossing Rapid
Lesser time for
First and most critical step immunohistochemistry and in-
Aim: to prevent decay and preserve situ hybridization
cells and tissues in a “life-like” Disadvantages:
state. Stops enzyme activity, killing Penetrates 10-15mm only
microorganisms and hardening specimen No significant cross linking
while maintaining sufficient of the of protein molecules;
molecular structure to enable subsequent chemical fixation
appropriate staining methods to be may be needed
applied
Inactivation of lysosomal Factors involved in fixation:
hydrolytic enzymes – post-mortem Hydrogen ion concentration
decomposition (autolysis); or by Temperature
chemically altering,
Thickness of section
stabilizing, and making tissue
components insoluble Concentration
Reducing the risk of infection Duration of fixation
by preventing putrefaction after
death (bacterial/fungal Effects of fixatives:
colonization & overgrowth) Harden/soft and friable tissue
Goal: to harden and protect the tissue Makes cell resistant to damage
from trauma of further handling and distortion
Ideal volume of fixative is 10 Inhibit bacterial decomposition
to 20 times greater than the Increase optical differentiation
size or volume of the specimen of cells and tissue components
Promotes staining 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 Schaudinn’s fixative
For preservation and storage of Alcohol-containing mercury
surgical, post-mortem and fixative
research specimens Used for wet smear preparation
*best fixative for Fe pigments, and connective tissues
elastic fibers
CHROMATE FIXATIVES
10% formol saline
Penetrates and fixes tissues Chromic Acid
well, minimum shrinkage & Precipitates all CHON and
distortion, does not overharden adequately fixes carbohydrates
tissues Potassium Dichromate (K2Cr2O7)
Slow (>24 h) Preserves lipid and mitochondria
Regaud’s (Muller’s) Fluid (3%) K2Cr2o7
Metallic Fixative Recommended for demonstration of
A. Mercury Containing chromatin, mitochondria, mitotic
Most common metallic figures, golgi bodies, RBC and
fixative colloid containing tissue
Routine fixative of choice Orth’s Fluid (2.5% K2Cr2O7)
for preservation of cell Recommended for study of early
detail in tissue degenerative processes and
photography tissue necrosis
Recommended for renal
tissues, fibrin, CT, LEAD CONTAINING
muscles
Disadvantages: hardens Lillie’s Fixative – used for
outer layers only, black preservation of glycogen
granular deposits formed, mucopolysaccharide, and amyloid
corrosive to metals *fixes connective tissue mucin
Zenker’s Fluid PICRIC ACID FIXATIVES
Recommended for fixing small A. Picric Acid
pieces of liver, spleen, Preserves glycogen well but
connective tissue fibers, and causes considerable tissue
nuclei shrinkage
Good general fixative for Allows brilliant staining
adequate preservation of all B. Bouin’s Solution
kinds of tissue and gives Recommended for fixation of
excellent staining result embryos and pituitary
Zenker Formol (Helly’s Solution) biopsiy
An excellent microanatomic For embryos, glycogen, not
fixative for pituitary gland, BM good for kidneys,
& b calllood containing tissue mitochondria, hemolyzes RBC
such as the liver and spleen C. Brasil’s Alcoholic Picroformol
Made up of HgCI2 and Fixative
formaldehyde Excellent fixative for
Heidenhain’s Susa Solution glycogen
Recommended for tumor biopsy *highly explosive when dry
Made up of MGCI12, glacial
acetic acid and formalin Osmium Tetroxide (Osmic Acid)
B-5 Fixative Preserves cytoplasmic structures
Commonly used for BM biopsy well
HgCI12, anhydrous Na acetate Used extensively for
neurological tissues
Fixes fats
Inhibits hematoxylin Newcomer’s Fluid
Extremely volatile Recommended for fixing
mucopolysaccharides and nuclear
Flemming’s Solution protein
Most common chrome-osmium acetic Acts both as nuclear and
acid and fixative histochemical fixative
Excellent fixative for nuclear
structure FACTOR AFFECTING FIXATION
Flemming’s Solution without
Retarded by:
Acetic Acid
Size and thickness
Recommended for cytoplasmic
structures Presence of mucus
Presence of fats
ALCOHOL FIXATIVES Presence of blood
Cold temperature
Methyl Alcohol Enhanced by:
Excellent for fixing dry and wet Size and thickness
smears, blood smears and bone Agitation
marrow tissues
Moderate heat (37 to 56 degrees
Ethyl Alcohol
C)
Used at concentrations of 70-
100% Difficulties caused by improper
Lower concentration causes RBC’s fixation:
to be hemolyzed and WBC’s are
inadequately preserved Failure to arrest early
Gendre’s Fixative autolysis of cells: failure to
Sputum fix immediately (the tissue was
Made up of alcoholic formalin probably allowed to dry before
fixing); insufficient fixative
Removal of substances soluble in
OTHER FIXATIVES fixing agent: wrong choice of
fixative
Acetone Presence of artifact pigments on
It is used in fixing brain tissue sections: incomplete
tissues for the diagnosis of washing of fixative
rabies Tissues are soft and feather-
Dissolves fat, evaporates like in consistency: incomplete
rapidly, preserves glycogen fixation
poorly Loss or inactivation of enzymes
Heat Fixation needed for study: wrong choice
Involves thermal coagulation of of fixative
tissue proteins Shrinkage and swelling of cells
Usually employed for frozen and tissue structure:
tissue sections and preparation overfixation
of bacteriologic smear Tissue blocks are brittle and
Carnoy’s Fluid hard: prolonged fixation
Recommended for fixing
chromosomes, lymph glands, and INCOMPLETE FIXATION
urgent biopsy Results in:
Made up of ethyl alcohol, o Separation of tissue
glacial acetic acid and components on the flotation
chloroform 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