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