San Pedro College

Department of Medical Laboratory Science

MANUAL OF HISTOPATHOLOGIC TECHNIQUES

Learning Activity No. 3

The Proper Fixation of Tissue Samples

SPECIFIC LEARNING OBJECTIVES:

At the end of the activity, the Medical Laboratory Science student should
be able to:
1. Explain the theoretical principles of fixation.
2. Identify the factors that enhance or retard the process of fixation.
3. Identify the different types of fixatives.
4. Appreciate the advantages of each type of fixative.
5. Achieve a keen awareness of the limitations of each type of fixative.
6. Value the relevance of adequate fixation in achieving accurate diagnosis.
7. Perform a supervised practice of the fixation technique.

LEARNING CONTENT

Fixation is the first and most critical step, the basis of histological
techniques. It is the process by which the cell and tissue constituents are
preserved with the least alteration from the living state with the use of fixatives.
It also prevents the breaking down of cellular elements via autolysis by
inactivating lysosomal enzymes, making the tissue components insoluble. In
addition, fixatives must also render the specimen insensitive to subsequent
treatment as may be necessary to permit microscopic examination.

Since the result of all subsequent procedures depend on the selection and
correct use of fixatives, it is, therefore, essential to understand the action of
fixatives upon the cell and tissue constituents in order to produce a tissue
section of good quality that will enable the pathologist to render a proper
diagnosis.

Factors Affecting Fixation

There are a number of factors that will influence the rate and effectiveness
of tissue fixation. These include:

A. pH – The process should be kept in the physiological range, between pH

6-8. The pH for the ultrastructure preservation should be buffered between
7.2 to 7.4. 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, a brown-black granular artefact
pigment which is deposited in blood-rich tissues. This pigment is a nuisance
as it can be confused with microorganisms or other pathological pigments.
Although the pigment can be removed from sections with saturated
aqueous picric acid before staining, it is preferable to avoid its formation in
the first place. For this reason and because formaldehyde reacts most
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 effectively at about a neutral pH, 10% formalin solutions are usually buffered
to pH 6.8 – 7.2.

B. Osmolality - The osmotic effects exerted by the fixative are again 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. Generally, it is
the osmolality of the vehicle (buffer) that is most important and in some
formulations this is adjusted to resemble that of tissue fluid (eg. formalin in
isotonic saline). Before fixation occurs cells can certainly be damaged by
non-isotonic fluids such as water and if specimens cannot be immediately
fixed they can be kept moist with gauze soaked in isotonic saline for a short
time. It is not a good idea to hold tissue immersed in saline for extended
periods.

C. Size of the Specimen - A specimen should not be more than 4 mm thick.

Ideally a 3 mm thick slice should provide excellent fixation and processing.
It is useful to remember that the specimen cavity in a standard processing
cassette is 5 mm deep.

D. Volume of the Fixative - A fixative to tissue ratio of 20:1 is considered the

lowest acceptable ratio. It is important to have an excess volume of fixative
in relation to the total volume of tissue because with additive fixatives the
effective concentration of reagent is depleted as fixation proceeds and in
a small total volume this could have an effect on fixation quality.

E. Temperature - Increasing the temperature of fixation will increase the rate

of diffusion of the fixative into the tissue and speed up the rate of chemical
reaction between the fixative and tissue elements. It can also potentially
increase the rate of tissue degeneration in unfixed areas of the specimen.
Fixation is routinely carried out at room temperature. Microwave fixation
may involve the use of higher temperatures, up to 65°C, but for relatively
short periods.

F. Time - The optimal time for fixation will vary between fixatives. For fixation to
occur, the fixative has to penetrate, by diffusion, to the centre of the
specimen and then sufficient time has to be allowed for the reactions of
fixation to occur. Both diffusion time and reaction time depend on the
particular reagent used and the optimum time will vary from fixative to
fixative. Formalin fixation is usually about 24 hours.

G. Penetration rate - The penetration rate of a fixing agent depends on its

diffusion characteristics and varies from agent to agent. As a general rule,
the penetration rate of formalin is 1hour per 1mm.
Type Of Fixatives

ALDEHYDES

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 Aldehyde 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.

Formaldehyde is the only gaseous aldehyde and is dissolved in water to

saturation at 37% – 40% w/v. This solution is generally referred to as “formalin”
or “concentrated formaldehyde solution”. For fixation, one part formalin is
usually diluted with nine parts of water or buffer. This produces a 10% formalin
solution which contains about 4% formaldehyde w/v, an optimal
concentration for fixation. Paraformaldehyde, a highly polymerised form of
formaldehyde may be deposited as a white precipitate in concentrated
formaldehyde solutions. To prevent this, small quantities of methanol (up to
15%) are commonly added to proprietary solutions.

There are well known hazards associated with the use of formaldehyde as
a fixative through skin or eye contact or via the respiratory tract. It is irritant,
corrosive and may cause allergic sensitization. Studies have shown that
formaldehyde causes nasopharangeal cancer, sinonasal cancer and myeloid
leukaemia. For these reasons in most countries there are strict guidelines in
place to limit the exposure of workers to formaldehyde in the workplace.

Glutaraldehyde acts similar to formaldehyde, however glutaraldehyde 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. One of the advantages of glutaraldehyde 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 4ºC,
and gives best overall cytoplasmic and nuclear detail. However it is not ideal
for immunohistochemistry staining. For electron microscopy glutaraldehyde
primary fixation is commonly followed by secondary fixation in osmium
tetroxide. Glutaraldehyde is not normally used for routine histopathology.

Some fixation protocols call for a combination of formaldehyde and

glutaraldehyde 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.

ALCOHOLS

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.

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. Ethanol is
sometimes used to preserve glycogen but will cause distortion of nuclear and
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cytoplasmic detail. 95% ethanol is used as a fixative for cytology smears.
Methanol is commonly used as a fixative for blood films. Both alcohols are
usually combined with other reagents when used as fixatives for tissue
specimens.

ACETIC ACID

Acetic acid 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.

OSMIUM TETROXIDE

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. The most important fixation reactions of osmium tetroxide are those
involving unsaturated bonds of lipids and phospholipids as it is one of the few
fixatives that stabilises lipids.

During fixation osmium tetroxide is reduced to lower oxides which are black
and insoluble and these are deposited in tissues, particularly on membranes.
Because osmium 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 lipids (particularly myelinated nerve fibres) at the
light microscope level. Generally it is used at about 1% w/v as a specialised
secondary fixative for electron microscopy and for teased nerve fibre
preparations.

CHROMATES

Potassium dichromate is a component of several compound fixatives

(e.g. Zenker’s and Helly’s solutions). The fixation reactions are thought to
involve the oxidation of proteins with the interaction of reduced chromate ions
forming some cross-links, the extent being determined by the pH of the fixative.
Chromium ions are reported to react with carboxyl and hydroxyl side chains of
proteins. It leaves amino groups available and thus favours staining with acid
dyes. Chromate will react with unsaturated lipids rendering them insoluble, it
being for this reason that it is regarded as a good fixative for mitochondria. It
is normally recommended that tissues fixed in a chromate containing fixative
are thoroughly washed in water prior to processing to avoid the formation of
an insoluble chromate sub-oxide by reaction of processing alcohol and the
chromate salt. Traditionally dichromate containing fixatives were used in
histochemical methods for “chromaffin” granules of endocrine tissues.

MERCURIC CHLORIDE

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 It is a powerful protein coagulant which leaves tissue in a state which
produces strong staining with acid dyes. 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.

There are several disadvantages to using fixatives containing mercuric

chloride. Apart from the corrosive nature of mercuric chloride, mercury is highly
toxic, can be absorbed through the skin and is a cumulative poison. During
fixation with fixatives containing mercuric chloride a crystalline or amorphous
greenish-brown artefact pigment of mercury is randomly deposited in tissues.
Treatment of specimens with Lugol’s iodine during processing or sections prior
to staining, will produce mercuric iodide which can be washed out of the
tissues. A subsequent treatment with sodium thiosulphate removes residual
iodine. Mercuric chloride-based fixatives tend to penetrate poorly and if
fixation is prolonged tissues become very hard and are prone to shrinkage
during processing.

PICRATES

Picric acid 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. For fixation, it is always used in combination
with other agents. It imparts a yellow color to tissues during fixation and
because of its acidic nature residual picric acid should be washed from tissues
with 70% ethanol before processing. If residual picric acid remains in tissue
blocks after processing, the staining characteristics of the tissue will be
affected and will deteriorate in time.

Picric acid appears not to fix lipids or most carbohydrates but is

recommended as a component in fixatives used to preserve glycogen. Picric
acid can hydrolyze nucleic acids so it should be avoided if DNA or RNA are to
be demonstrated. It will dissolve small deposits of calcium in specimens and a
considerable amount of shrinkage occurs during the processing of tissues fixed
in picric acid containing reagents.

ACETONE

Acetone 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. It is widely recommended for fixation as part of the
histochemical demonstration of enzymes where it is generally used cold (4°C).
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.

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TIME ALLOTMENT: 3 hours

LEARNING RESOURCES:

500ml beaker Scalpel/knife

Foil Tissue cassettes
10% formalin Gauze
Tissue samples A pair of scissors
Chopping board string

LEARNING STRATEGIES: Pre-lab discussion, Instructor-guided performance

PROCEDURE

1. Examine the tissue grossly. Note its color, texture, size, consistency and any
remarkable characteristics displayed by the tissue.
2. Using a scalpel or knife, cut a representative specimen into 2 cm2 but not
more than 4mm in thickness.
3. Place the specimen in a tissue cassette. Write your name (or code) and
attach it inside the cassette. You may also opt to label the plastic cassette
with a pencil instead of attaching the piece of paper.
4. Fill a 500ml beaker with 10% formalin up to the 400ml mark. Submerge the
tissue-filled cassette into the solution.
5. Cover the beaker with foil. Label it with your group number and section.
Send it to the laboratory for storage until the next laboratory session.

LEARNING ACTIVITY NO. 3

STUDENT LEARNING EVALUATION

The Proper Fixation of Tissue Samples

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Name : _______________________ Score: ________
Course/Yr./Sec.:_______________ Date: _________

Guide Questions:

1. How can the following factors accelerate or speed up fixation

time?(10pts)
a. pH ________________________________________________
________________________________________________
b. temperature ________________________________________________
________________________________________________
c. size of sample ________________________________________________
________________________________________________
d. fixative volume ________________________________________________
________________________________________________
e. agitation ________________________________________________
________________________________________________

2. What is the significance of osmolality in the fixation process? (10pts)

_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________

3. Identify the advantages and disadvantages of the fixatives listed in the

table below. Fill in your answers to complete the table. (10pts)

FIXATIVE ADVANTAGE (1pt each) DISADVANTAGE (1pt each)

Formaldehyde

Mercuric Chloride

Osmium tetroxide

Acetone

Picric acid

Rubrics for Scoring Guide Questions

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 10 pts 8 pts 6pts 4 pts 1 pts

Excellent Good Average Fair Needs

The answer The answer The answer The answer improvement
meets all of meets most of meets some of meets only a The answer
the criteria: the criteria: the criteria: few of the does not meet
1.Relevance 1. Relevance 1. Relevance criteria: any of the
2. Clarity 2. Clarity 2. Clarity 1. Relevance following
3.Conciseness 3.Conciseness 3.Conciseness 2. Clarity criteria:
4. Grammar 4. Grammar 4. Grammar 3.Conciseness 1. Relevance
4. Grammar 2. Clarity
3. Conciseness
4. Grammar

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