FIXATION Flashcards
• The first & most critical step; Basis of histological techniques.
FIXATION
• It is done after the process of gross examination.
FIXATION
It is the method of preserving cells & tissues in life-like states as possible (it is the first crucial stage in tissue processing).
Fixation
• It is the process by which the cell & tissue constituents are preserved with the least alteration from the living state with the use of fixatives.
Fixation
prevents the breaking down of cellular elements via autolysis by inactivating lysosomal enzymes, making the tissue components insoluble.
Fixation
must render the specimen insensitive to subsequent treatment as may be necessary to permit microscopic examination.
• Fixatives
Factors Affecting Fixation
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Penetration rate
Volume of the fixaitve
Osmolality
Temperature
Time
Size of the specimen
Types of Fixatives
Aldehyde
Alcohols
Acetic acid
Acetone
Chromates
Osmium tetroxide
Mercuric chloride
Picrates
pH
The process should be kept in the physiological range, between the
pH: 6 to 8 (neutral pH)
pH
The pH for the ultrastructure preservation should be buffered between
7.2 to 7.4.
is the most commonly used fixative in the histopathology laboratory.
formalin
Unbuffered formalin will slowly oxidize to formic acid resulting in a fall in pH.
Under these conditions, the formic acid will react with hemoglobin, forming:
Acid formaldehyde hematin
The osmotic effects exerted by the fixative are more important at the ultrastructural level than at the level of the light microscope because it is the phospholipid membranes that are easily damaged by excessively________
hypotonic or hypertonic solutions.
osmolality of the vehicle (____)
buffer
It is the ______ of the vehicle (buffer) that is most important & in some formulations, this is adjusted to resemble that of tissue fluid (e.g., formalin is isotonic saline).
osmolality
Before fixation occurs, cells can certainly be damaged by non-isotonic fluids such as water & if specimens cannot be immediately fixed, they can be kept moist with gauze soaked in __________ for a short time.
isotonic saline
Size
A specimen should not be more than_____ thick.
4mm
Size
Ideally, a_____ thick slice should provide excellent fixation & processing.
3mm
Size
It is useful to remember that the specimen cavity in a standard processing cassette is____ deep.
5mm
Volume of fixative
A fixative-to-tissue ratio of _____is considered the lowest acceptable ratio.
20:1
Temperature
Increasing the temperature of_____ will increase the________ of the fixative into the tissue & speed up the rate of______between the fixative & tissue elements.
fixation
rate of diffusion
chemical reaction
It can also potentially increase the rate of tissue degeneration in unfixed areas of the specimen.
Temperature
Fixation is routinely carried out at_____ temperature (recommended temperature).
room
may involve the use of higher temperatures (up to 65°C), but for relatively short periods.
Microwave fixation
Microwave fixation may involve the use of higher temperatures (up to_____), but for relatively short periods.
65°C
T or F
The optimal time for fixation will vary between fixatives.
True
For fixation to occur, the fixative has to______, by diffusion, to the center of the specimen and then sufficient time has to be allowed for the reactions of fixation to occur.
penetrate
Formalin fixation usually takes up about ____
24 hours
_______of a fixing agent depends on its diffusion characteristics & varies from agent to agent.
penetration rate
penetration rate of formalin is_____
1 mm/hr.
act by creating covalent chemical bonds between proteins in tissues.
Aldehyde fixatives
This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue.
Aldehyde
Different types of aldehyde include:
- Formaldehyde
- Glutaraldehyde
Formaldehyde
The only gaseous aldehyde & is
dissolved in water to saturation at
37% to 40% w/v.
This solution is generally referred to as “formalin” or “concentrated formaldehyde solution”.
37% to 40% w/v.
For fixation, 1 part formalin is usually diluted with___ parts of water or buffer.
9
, a highly polymerized form of formaldehyde may be deposited as a white precipitate in concentrated formaldehyde solutions.
Paraformaldehyde
FIXATION: PROCEDURE
Examine the tissue grossly.
Cut a representative specimen into 2cm2, but not more than 4mm in thickness.
Place the specimen inside the cassette. You may also opt to label the plastic cassette with a pencil instead of attaching the piece of paper.
Fill a 500mL beaker w/ 10% formalin up to the 400mL mark. Submerge the tissue-filled cassette into the solution.
Cover the beaker with foil. Label it with your group number & section. Send it to the laboratory for storage until the next laboratory session.
Unbuffered formalin will slowly oxidize to———— resulting in a
fall in pH.
Under these conditions the formic acid will react with hemoglobin
forming————-, a brown-black granular artefact pigment which is deposited in blood-rich tissues.
formic acid
acid formaldehyde hematin
formaldehyde reacts most effectively at about a neutral pH, 10% formalin solutions are usually buffered to
pH 6.8 – 7.2.
Studies have shown that
——— causes nasopharangeal cancer, sinonasal cancer and myeloid leukaemia.
For these reasons in most countries there are strict guidelines innplace to limit the exposure of workers to ——— in the workplace
formaldehyde
is a larger molecule, and so its rate of diffusion across membranes is slower.
It is recommended that tissue be less than 1mm in thickness in at least one
dimension.
glutaraldehyde
One of the advantages of———- fixation is that it may offer a more rigid or tightly linked fixed product.
It causes rapid and irreversible changes, fixes quickly, is well suited for electron microscopy, fixes well at——, and gives best overall cytoplasmic and nuclear detail.
However it is not ideal for———— staining.
glutaraldehyde
4ºC
immunohistochemistry
For electron microscopy glutaraldehyde
primary fixation is commonly followed by secondary fixation in——-
osmium tetroxide
Some fixation protocols call for a combination of———-&———- so that their respective strengths complement one another.
These crosslinking fixatives–especially formaldehyde–tend to preserve the
secondary structure of proteins and may protect significant amounts of tertiary
structure as well.
formaldehyde and
glutaraldehyde
Precipitating (or denaturing) fixatives act by reducing the solubility of
protein molecules and (often) by disrupting the hydrophobic interactions that give many proteins their tertiary structure.
The precipitation and aggregation of proteins is a very different process from the crosslinking that occurs with the aldehyde fixatives.
Alcohol
The most common precipitating fixatives are——-&——-
ethanol and methanol
They are commonly used to fix frozen sections and smears.
Fixation commences at
a concentration of 50 – 60% for ethanol and >80% for methanol.
They are commonly used to fix frozen sections and smears.
Fixation commences at
a concentration of 50 – 60% for ethanol and >80% for methanol.
is sometimes used to preserve glycogen but will cause distortion of nuclear and cytoplasmic detail.
Ethanol
———% is used as a fixative for cytology smears.
95% ethanol
———% is used as a fixative for cytology smears.
95% ethanol
is commonly used as a fixative for blood films. Both alcohols are
usually combined with other reagents when used as fixatives for tissue
specimens.
Methanol
is coagulant in action with nucleic acids but generally does not fix proteins.
It is incorporated in compound fixatives to help prevent the loss of nucleic acids and, because it swells collagen, to counter the shrinkage caused by other ingredients such as ethanol.
Acetic acid
penetrates very rapidly but fixatives that contain it will lyse red blood cells.
Acetic acid
It is a highly toxic, volatile, crystalline solid which is supplied in sealed ampoules.
Because of its volatility it must be handled with great care under a fume hood because it will readily fix the conjunctiva of the eye and the nasal mucosa.
OSMIUM TETROXIDE
The most important fixation reactions of_______ are those involving unsaturated bonds of lipids and phospholipids as it is one of the few fixatives that stabilises lipids
osmium tetroxide
During fixation _______is reduced to lower oxides which are black and insoluble and these are deposited in tissues, particularly on membranes.
osmium tetroxide
_______ is a heavy metal it scatters electrons and thus adds electron density to the electron microscope image.
It can also be used as an en bloc stain for demonstrating_____ (particularly myelinated nerve fibres) at the light microscope level.
osmium
lipids
Generally it is used at about 1% w/v as a specialised secondary fixative for electron microscopy and for teased nerve fibre preparations.
Osmium
It is a powerful protein coagulant which leaves tissue in a state which produces strong staining with acid dyes.
Mercuric chloride
It reacts with phosphate residues of nucleic acids and effectively fixes nucleoproteins.
It is for this reason that it is the major component in formulated fixatives such as B-5 and Helly’s, fixatives recommended when high quality nuclear preservation is required
Mercuric chloride
Apart from the corrosive nature of________, it is highly toxic, can be absorbed through the skin and is a cumulative poison
mercuric chloride, mercury
During fixation with fixatives containing_______ a crystalline or amorphous greenish-brown artefact pigment of _____ is randomly deposited in tissues.
mercuric chloride; mercuric chloride
Treatment of specimens with______ during processing or sections prior to staining, will produce mercuric iodide which can be washed out of the tissues.
A subsequent treatment with_______ removes residual iodine.
Lugol’s iodine
sodium thiosulphate
_______-based fixatives tend to penetrate poorly and if fixation is prolonged tissues become very hard and are prone to shrinkage during processing.
Mercuric chloride
is a bright yellow crystalline substance that must be stored wet with water to avoid the risk of explosion by percussion or heating of the dry substance
It should be kept in a tightly sealed container and regularly dampened with distilled water
Picric acid
For fixation, it is always used in combination with other agents.
It imparts a______ color to tissues during fixation and because of its acidic nature residual picric acid should be washed from tissues with______ before processing.
yellow
70% ethanol
Picric acid appears not to fix____ or most_______ but is recommended as a component in fixatives used to preserve______.
lipids
carbohydrates
glycogen
Picric acid can hydrolyze nucleic acids so it should be avoided if ________are to be demonstrated.
It will dissolve small deposits of____ in specimens and a considerable amount of shrinkage occurs during the processing of tissues fixed in picric acid containing reagents.
DNA or RNA
calcium
has a similar action to alcohol and has been used as a fixative and dehydrant for tissue processing, particularly rapid hand-processing of small specimens
Acetone
It is widely recommended for fixation as part of the histochemical demonstration of enzymes where it is generally used cold (4°C).
Acetone
It is an effective lipid solvent with a rapid action which can make tissues very brittle. Because it is highly volatile and flammable it is generally not used on automatic tissue processors
Acetone
