Week 4: Fixations Flashcards
This is done to
prevent the decomposition and drying
of tissue samples so that it can be
processed in the laboratory.
Fixation
This process is also considered
the first and most critical step
in histotechnology.
Fixation
o Heating (blue flame),
microwaving, cryopreservation
(freeze-drying)
Physical fixation
o Achieved by immersing the specimen in a fixative or by perfusing the vascular system with fixative o For specialized histochemical procedures, fixatives can be applied in vapor form
Chemical fixation
Fixatives chemically link/add
themselves on to the tissue
giving stability to the protein
Additive fixation
Example of additive fixation
formalin, mercury, osmium
tetroxide
Example of non-additive fixation
acetone, alcohols
Fixation ph
6-8
What happens in tissue hypoxia?
Lower the pH of the solution
What temperature is sufficient to maintain excellent
morphology of tissues
room temperature
What temperature is used for regular tissue processing
40°C
What temperature is used for electron microscopy and
some histochemistry
0-4°C
What fixative is used for osmolarity?
Normal phosphate buffered
saline (PBS)
Concentration of fixative in routine laboratory
10% formaldehyde
Concentration of fixative in immune electron microscopy
3%
glutaraldehyde, 0.25%
glutaraldehyde
commonly used in
routine laboratory
formalin
Made up of only one component substance (e.g. Formaldehyde, HgCl, acetone, alcohol, glutaraldehyde)
Simple
Made up of two or more
fixatives which have been
added together
Compound
Example: Helly’s solution (made up of formaldehyde, mercurric chloride and potassium dichromate, Gendre’s fixative (ethyl alcohol, formaldehyde, glacial acetic acid)
Compound
Permits general microscopic study of tissue structures without altering the structural pattern and normal (e.g. 10% formol saline, Heidenhain’s Susa)
Microanatomical fixatives
Preserves specific
elements of the cell
Cytological fixatives
Failure to arrest early
autolysis of cells
Failure to arrest early
autolysis of cells
Removal of substance
soluble in fixing agent
Wrong choice of fixative
Presence of artefact
pigments on tissue
sections
Incomplete washing of
fixative
Tissues are soft and
feather-like in consistency
Incomplete fixatio
Loss or inactivation of
enzymes needed for study
Wrong choice of fixative
Shrinkage and swelling of
cells and tissue structure
Over fixation
Tissue blocks are brittle
and hard
Prolonged fixation
is the process of
placing an already fixed tissue in a
second fixative in order
Secondary fixation
is a form of secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5-3% potassium dichromate for 24 hours to act as mordant for better staining effects and to aid in cytologic preservation of tissues
Post-Chromatization
It is a polymerized form of formaldehyde, usually obtained as a fine white powder, which depolymerizes back to formalin when heated
Paraformaldehyde
It is suitable for paraffin embedding
and sectioning, and also for
immunocytochemical analysis
Paraformaldehyde
It is a dialdehyde – made up of two
formaldehydes
Glutaraldehyde
➢ Most frequently used for the fixation
of specimens for electron microscopy
➢ But not good for immunohistochemical
staining
Glutaraldehyde
10% formal-saline
Aldehyde
suitable blood films or cell
cultures
Methanol
very good for
cytologic smears
o Example: PAP smears
Alcohols
It is excellent for fixing dry and
wet smears, blood smears and
bone marrow tissues
100% Methyl Alcohol
It is used at concentrations of
70-100%
Ethyl Alcohol
It is considered to be the most rapid fixative and may be used for urgent biopsy specimens for paraffin processing within 5 hours
Carnoy’s Fixative
used
on frozen sections and smears
Clarke’s solution
o It can be used for rapid diagnosis because it fixes and dehydrates at the same time, e.g., in the frozen section room o It is good for preservation of glycogen
Gendre’s Fixative
This is recommended for the
fixing of mucopolysaccharides
and nuclear proteins
Newcomer’s Fluid
