Transcript Fixation

To be able to differentiate histotechnology
and histotechnologist.
To be able to differentiate the advantages
and disadvantages of fresh tissue examination.
To be able to know the basis for method
used in
fresh tissue examination.
To be able to name and differentiate the
fresh tissue examination.
To be able to understand the fresh frozen
Learning Objectives:
The tissue may be done fresh or preserved.
Advantage of fresh tissue examination: The specimen
may be in living state, therefore may observe protoplasmic
activities (motility, mitosis, phagocytosis, pinocytosis).
Disadvantage: not permanent, and therefore liable to changes
Histotechnology - is the art and science performed by
the histotechnologist to produce a tissue section of good
quality that will enable the pathologist to diagnose the presence
of disease.
Cell Structure and chemical components
Amount and nature of tissue
Urgency of result
The choice of tissue examination method would depend
on the following conditions;
The methods of fresh tissue examination:
1. Teasing or dissociation – selected tissue is in isotonic solution,
dissected and examined under the microscope.
2. Squash Preparation (Crushing) – less than 1 mm tissue pieces
are compressed (stained when necessary), and examined
the microscope.
3. Smear preparation – spread cellular materials (secretions,
sediments) are examined. useful for cytologic examinations
a) Streaking – using an applicator stick by direct or zigzag
b) Spreading – teasing (sputum, bronchial aspirates, thick
mucoid secretions) by applicator stick and spread in
circular spread.
c) Pull-Apart – thick secretions (gastric lavage, serous fluids,
blood) are dispersed evenly on two slide surfaces.
d) Touch Preparation (Impression) - freshly cut tissue surface
is brought into contact to slide. Cells are examined in their
actual intercellular relationship.
4. Frozen Section – for rapid diagnosis during surgery, and
demonstration of lipids and nervous tissue elements.
Frozen tissue – 10 -15 u in thickness cut from frozen tissue on a microtome with CO2 or on cryostat.
A cryostat is cold chamber kept at an atmospheric temperature of -10 to -20 deg. C. The frozen
sections are then transferred to a slide, and examined under light microscopy.
Sequence of fresh frozen tissue examination by cryostat
The tissue should be fresh and processed quickly. Slow freezing can cause tissue distortion due to
ice artifacts. The more commonly used methods of freezing include;
Liquid nitrogen
Isopentane cooled by liquid nitrogen
Carbon dioxide gas
Aerosol sprays
Chapter 2:
Fixation and Fixatives
To understand the process of autolysis
To determine the contributing factors in autolysis.
To discuss why certain tissues are affected severely by autolysis.
To know the objectives of fixation and qualities to serve its objective.
To understand the factors involve in fixation.
To know the types of fixative.
To understand the properties of formaldehyde.
Learning objectives:
Fixation process
• Fixation is usually the
first stage in a multistep
process to prepare a
sample of biological
material for microscopy
or other analysis.
• Therefore, the choice of fixative
and fixation protocol may depend
on the additional processing steps
and final analyses that are planned.
• For example,
immunohistochemistry utilizes
antibodies which bind to a specific
protein target.
• Prolonged fixation can chemically
mask these targets and prevent
antibody binding. In these cases, a
'quick fix' method using cold
formalin for around 24 hours is
typically used.
Chapter 2:Fixation and Fixatives
Once tissues are removed from the body, they undergo a
process of self-destruction or autolysis. Soon after tissue death, the
intracellular enzymes break down the protein and then the cell
eventually undergo liquefaction. – Autolysis.
Properties of Autolysis:
independent of a bacterial action
retarded by cold
accelerated at 30 degree C temperature, inhibited at 50 degrees
more severe in tissue which are rich in enzymes (e.g. liver, brain, and
kidney) and less rapid in elastic and collagen tissues.
Chapter 2:Fixation and Fixatives
“washed-out” appearance with swelling of cytoplasm,
eventually to a granular, homogenous mass which fails to take
up stains.
The nuclei of autolytic cells may show some of the changes
including condensation (pyknosis), fragmentation
(karyorrhexis) and lysis (karyolysis).
There may be diffusion of intracellular importance like
glycogen. The epithelium may desquamate from its basement
Dead tissue may allow bacteria to proliferate. When these
bacteria decompose, it can mimic those of autolyzed tissue.
Autolysed tissue presentations:
Chapter 2:Fixation and Fixatives
Fixative should have the following qualifications to serve its objective;
a) Arrest autolysis and bacterial decomposition
b) Stabilizes cellular and tissue constituents.
c) Preservation of tissue substances and proteins (for
Immunohistochemisatry techniques to augment diagnosis)
Fixation is therefore the first step and the foundation in a
sequence of events that culminate in the final examination of ta tissue
Each fixative has specific properties and disadvantages. Their
varied effects to tissues would require careful selection of fixative
when specific cellular substances is to be studied.
The objective of fixation;
To preserve cells and tissue constituents in as close to life-like
To ready for further processing without or minimal tissue change.
In the fields of histology,
pathology, and cell biology,
fixation is a chemical process
by which biological tissues are
preserved from decay, either
through autolysis or
• Fixation terminates any
ongoing biochemical
reactions, and may also
increase the mechanical
strength or stability of the
treated tissues.
• Purposes of fixation
1- To prevent or arrest autolysis
and bacterial decomposition
and putrefaction.
2- To coagulate the tissues as to
prevent loss of easily diffusible
3-To fortify the tissue against
the deleterious effects of the
various stages of tissue
4- To leave the tissues in a
condition which facilitate
differential staining with dyes
and other reagents
Chapter 2:Fixation and Fixatives
2. Size of specimen – the penetration of fixative is inversely directly relative to size. In which, larger
tissue size will be fixed slowly. Thus, large specimens are opened and washed of contaminant or sliced
thinly. . The fixative should be at least 20 times the volume of the specimen. Cut sections should be 14 mm Thickness
3. Change in volume – the volume changes in tissue with fixation, as the tissue shrinks by 33%. This is
evident as the tissue show larger nuclei and cells in frozen sections (unfixed).
4. pH and Buffers – The hydrogen ion concentration varies between fixatives, Should be kept in the
physiological range, between pH 4-9. The pH for the ultra structure preservation should be buffered
between 7.2 to 7.4
The buffer systems maintain this physiological range of pH level (e.g.; phosphate, acetate,
bicarbonate). The buffer should have the following properties;
a. do not interfere with fixative
b. do not inhibit enzymes
5. Osmolality – the fixative solutions; osmotic pressure may be affected by the addition of buffer. The
best result is obtained by using slightly hypertonic solutions (isotonic solutions adjusted by using
sucrose). Hypertonic solutions give rise to cell shrinkage. Hypotonic solutions result in cell swelling and
poor fixation.
6. Concentration of fixative
7. Duration of fixation - As a general rule 1hour per1mm
Factors involve in Fixation:
1. Temperature – standardized fixation is carried out at room temperature. However, for electron
microscopy and some histochemical procedure, fixation is usually at 0 – 4 degrees Celsius. Lower
temperatures autolysis is slowed down allowing a more life-like appearance of tissues.
Types of fixation
There are generally three types of fixation process:
Heat fixation:
After a smear has been allowed to
dry at room temperature, the slide is
gripped by tongs or a clothespin and
passed through the flame of a Bunsen
burner several times to heat-kill and
adhere the organism to the slide
2. Perfusion:
Fixation via blood flow. The fixative is injected
into the heart with the injection volume
matching cardiac output.
The fixative spreads through the entire body,
and the tissue doesn't die until it is fixed.
This has the advantage of preserving perfect
morphology, but the disadvantages that the
subject dies and the cost is high (because of
the volume of fixative needed for larger
3. Immersion:
The sample of tissue is immersed in
fixative of volume at a minimum of
20 times greater than the volume of
the tissue to be fixed.
The fixative must diffuse through
the tissue in order to fix, so tissue
size and density, as well as the type
of fixative must be taken into
Chemical Fixation
• Process whereby Structures are preserved in
a state (both chemically and structurally) as
close to living tissue as possible.
• This requires a chemical fixative which can
stabilize the proteins, nucleic acids and
mucosubstances of the tissue by making
them insoluble.
Types of Chemical Fixatives
1. Cross linking fixatives – ALDEHYDES
• Cross linking fixatives act by creating
covalent chemical bonds between proteins
in tissue. This anchors soluble proteins to the
cytoskeleton, and lends additional rigidity to
the tissue.
• By far the most commonly used
fixative in histology is
formaldehyde. It is usually used
as a 10% Neutral Buffered
Formalin (NBF).
• Formaldehyde is a gas. Formalin
is formaldehyde gas dissolved in
• Paraformaldehyde is a
polymerized form of
formaldehyde, usually obtained
as a fine white powder, which
depolymerises back to formalin
when heated.
• Formaldehyde fixes tissue by crosslinking the proteins, primarily the
residues of the basic amino acid
• Its effects are reversible by excess
water and it avoids formalin
pigmentation. Other benefits
include: Long term storage and
good tissue penetration.
• It is particularly good for
immunohistochemistry techniques.
• Also the formaldehyde vapour can
be used as a fixatives for cell
2. Glutaraldehyde.
- causing deformation of the alpha-helix
structures in proteins.
- larger molecule, and may not penetrate
thicker tissue specimens as effectively as
Thus small blocks of tissue are required.
- may offer a more rigid or tightly linked fixed
product—its greater length and two aldehyde
groups allow it to 'bridge' and link more distant
pairs of protein molecules.
• It causes rapid and irreversible
changes, fixes quickly, is good for
electron microscopy, fixes well at
4oC, and gives best overall
cytoplasmic and nuclear detail.
• However it is not ideal for
immunohistochemistry staining
• Some fixation protocols call for a
combination of formaldehyde and
glutaraldehyde, so that their
respective strengths complement
one another.
Precipitating fixatives : Alcohols
Precipitating (or denaturing) fixatives
• The most common precipitating
fixatives are ethanol and methanol.
They are commonly used to fix frozen
sections and smears.
- act by reducing the solubility of protein
• Acetone is also used and has been
shown to produce better histological
hydrophobic interactions which give many
preservation than frozen sections
when employed in the Acetone
proteins their tertiary structure.
Methylbenzoate Xylene (AMEX)
molecules and (often) by disrupting the
- The alcohols, by themselves, are known to • The protein denaturants - methanol,
cause considerable shrinkage and hardening ethanol and acetone - are rarely
used alone for fixing blocks unless
of tissue during fixation while acetic acid
studying nucleic acids.
• Acetic acid is a denaturant that is
alone is associated with tissue swelling;
sometimes used in combination with
combining the two may result in better
the other precipitating fixatives.
preservation of tissue morphology
Oxidising agents
The oxidising fixatives can react
with various side chains of
proteins and other
biomolecules, allowing the
formation of crosslinks which
stabilize tissue structure.
However they cause extensive
denaturation despite preserving
fine cell structure and are used
mainly as secondary fixatives.
• Osmium tetroxide is often
used as a secondary fixative
when samples are prepared
for electron microscopy. (It is
not used for light microscopy
as it penetrates thick sections
of tissue very poorly.)
• Potassium dichromate,
chromic acid, and potassium
permanganate all find use in
certain specific histological
Mercurials and Zenker's have an
unknown mechanism which increases
brightness of staining along with giving
excellent nuclear detail. Despite being
fast, mercurials penetrate poorly and
produce tissue shrinkage.
• Their best application is for fixation of
hematopoietic and reticuloendothelial
tissues. Also note that since they
contain mercury care must be taken
with disposal
Picrates penetrate tissue well to react
with histones and basic proteins in
order to form crystalline picrates with
amino acids and precipitate all proteins.
It is a good fixative for connective
tissue, preserves glycogen well and
extracts lipids in order to give
superior results to formaldehyde in
immunostaining of biogenic and
polypeptide hormones
• However it causes a loss of basophilia
unless the specimen is thoroughly
washed following fixation
• HOPE Fixative
Hepes-glutamic acid buffer-mediated
organic solvent protection effect (HOPE)
gives formalin-like morphology,
excellent preservation of protein
antigens for immunohistochemistry
and enzyme histochemistry, good RNA
and DNA yields.
Chapter 2:Fixation and Fixatives
A. Microanatomical fixatives – are those the permit the general microscopic study of tissues structures without
altering the structural pattern and normal intercellular relationship of the tissues in question.
10% formol saline
10% neutral buffered formalin
Zenkers solution
Bouin’s solution
B. Cytological fixatives – are those that preserve specific parts and particular microscopic elements of the cell
1. Nuclear fixative – are those that reserve nuclear structure (e.g. chromosome) in particular.
They usually contain glacial acetic acid as their primary component due to its affinity for nuclear chromatin.
Usually have pH of 4.6 or less
Heidenhain’s susan
2. Cytoplasmic – are those that preserve cytoplasmic structures in particular. They never contain
glacial acetic acid which destroys mitochondria and Golgi bodies of the cytoplasm. Ph of more than 4.6.
Kelly flemmings
Regauds’s fluid
Orth’s fluid
C. Histochemical fixative - preserve chemical constituents of cells and tissues.
Formol saline 10%
Absolute ethyl alcohol
Newcomer’s fluid
Types of fixative according to action:
Chapter 2:Fixation and Fixatives
1. The most widely used and routine fixative particularly for paraffin
embedded sections.
2. It is a gas with a very pungent odor.
3. The commercially available solution of formaldehyde (formalin)
contains 35 – 40% gas by weight. However, pure stock solution of
40% formalin is unsatisfactory.
4. Formaldehyde is commonly used as 4% solution, giving 10%
formalin for fixation. Therefore, it should be diluted 1:10.
5. Formaldehyde is usually buffered to pH 7 with phosphate buffer.
6. It is thought that formaldehydes form cross-links between proteins,
creating a gel, thus retaining cellular constituent.
7. It is a forgiving fixative – requires a relatively short fixation time (24
hours) but can be used for long term usage with no deleterious
effects on tissue.
8. Prepared by adding 100 ml of 40% formaldehyde to 900 ml distilled
water with 4 g sodium phosphatase (monobasic) and 6.5 g sodium
phosphate (dibasic anhydrous).
Formaldehyde (Formalin)
1. Formalaldehyde fixatives
• 10% formal saline
• Formalin 100 ml
• Sodium chloride 8.5 grams
• Tap water 900 ml
2. Buffered 10% formalin
Formalin 100 ml
Acid sodium phosphate
monohydrate 4 g
Anhydrous disodium phosphate
6.5 g
Tap water 900 ml
1. Carnoy’s fixatives
Absolute alcohol 60 ml
30 ml
Glacial acetic acid 10 ml
2. Picric acid fixatives
Bouin’s fluid
Rossman’s fluid
Gendre’s fluid
3. Mercuric chloride containing fixatives
Formal sublimate
Zenker’s solution
Susa fluid
Helly’s fluid