HISTOPATH LEC MIDTERMS

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FIXATION - PRIMARY GOAL OF FIXATION = preserve the

morphologic and chemical integrity of the cell in as life-like
TOPIC OUTLINE manner
1 Fixation • including
2 Methods of fixation o shape,
3 Benefits of Fixation o structure,
4 Practical Considerations o intercellular relationship
5 Main Factors o chemical constituents of tissues
6 Characteristics • stops all vital cellular processes to prevent
7 Types of Fixatives degeneration, decomposition, putrefaction, and
8 Secondary Fixation tissue distortion
9 General Precautions o no cellular processes → no changes
occur → “life-like” → primary goal
10 Difficulties Caused by Improper Fixation
• weak chemical associations are changed into
11 Fixation Artifacts
stable complexes that will not break down with
12 Lipid, Carbs, Protein Fixation
further histologic handling
13 Antigen Retrieval for Immunostaining
o weak chemical associations → stable
14 Microwave fixation complexes
15
- SECONDARY GOAL OF FIXATION = harden and
FIXATION protect the tissue from trauma of further handling
- 1st and most critical step in histotechnology • easier to cut and process for microscopy
• If fixation is not adequate, the other processes • more properly oriented in the cassette for paraffin
will also be inadequate embedding and microtomy
o dehydration,
o clearing, - fixation aims to prevent or arrest the degenerative
o infiltration, processes which commence as soon as a tissue is
o embedding, deprived of its blood supply
o microtomy and staining
• poorly processed tissue will make it difficult for - Loss & diffusion of soluble substances must be avoided
the pathologist to render a proper diagnosis through
o poorly processed tissue → no proper • precipitation
diagnosis • coagulation,
• cross-linking to other insoluble structural
- preserves tissues from decay preventing
components.
• autolysis
o tissue digestion by intracellular - The tissues must be largely protected against the
enzymes that are released when deleterious effects of tissue processing including
organelle membrane rupture infiltration with hot wax
• putrefaction / bacterial decomposition • retain their reactivity to stains and other reagents
o due to microorganisms in the specimen such as antibodies and nucleic acid probes
- Fixation should be carried out ASAP to prevent autolysis
• after removal of the tissue (surgical pathology) - changes that occurs in an aqueous environment
• soon after death (autopsy) • shrinkage
• swelling
- ACTION OF FIXATION
• hardening
• terminates ongoing biochemical reactions
• increase mechanical strength or stability of the - The tissues will undergo further changes during
treated tissues processing when they are placed in a non-aqueous
o done by immersing tissue sections or environment
smears in fixative fluid • Ex. 10% buffered formalin → slight swelling of
▪ SMEARS = drying acts as a tissue
form of preservation
• During processing, specimen may shrink & lose
• prevent autolysis by inactivating lysosomal 20-30% volume
enzymes
OBJECTIVES OF FIXATION
• stabilize the fine structure both inside and
1 To preserve tissue
between cells
2 To prevent breakdown of cellular elements
o done by making macromolecules
3 To coagulate or precipitate protoplasmic substances
resistant to dissolution by water and
other fluids 1. To Preserve the Tissue
• inhibit molds & bacteria growth that gives rise to - by stopping all cellular activities
putrefactive changes • Seen under the microscope as if still in their
- original living state

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-placed in appropriate fixative ASAP after removal from 2 MEANS OF FIXATION

the body A. PHYSICAL METHODS
- TYPES
- prolong exposure of tissue in • Heat Fixation
• Air = dry out • Microwave Fixation
o distortion in morphologic appearance • Cryopreservation (freeze-drying)
▪ water (hypotonic solution) = swell
▪ strong salt (hypertonic solution) = shrink A. HEAT FIXATION
2. To Prevent Breakdown of Cellular Elements - rarely used on tissue specimens
- Surgical removal of tissue from the body will deprive it of • Application: smears of microorganisms.
O2 & nutrition causing cell death
• Lysosome = "suicide sac" within the cytoplasm. B. MICROWAVE FIXATION
o Contains hydrolytic enzymes - widely practiced in routine laboratories
▪ Released when cell is • Form of heat fixation
destroyed → postmortem
decomposition or "autolysis" C. CRYO-PRESERVATION (freeze drying)
✓ Autolysis = occurs - some application in histochemistry
due to the action of • Not usually applied to diagnostic tissue
the hydrolytic specimens
enzymes B. CHEMICAL FIXATION
- TYPES
- Fixation prevent autolysis by: • Immersion fixation
• Inactivating lysosomal enzymes • Perfusion fixation
▪ chemically altering, stabilizing, and making the
• Vapor fixation
tissue components insoluble
A. IMMERSION FIXATION
.
- immersing the specimen in the fixative solution
- protects the tissue from further decomposition
("putrefaction") after death due to bacterial or fungal B. PERFUSION FIXATION
colonization and overgrowth. - perfusing or injecting the vascular system with fixative
3. To Coagulate or Precipitate Protoplasmic • Small animals or whole organs (ex. lungs)
Substances
- Fixation renders insoluble certain tissue components that C. VAPOR FIXATION
may otherwise leak out during subsequent histologic - for some specialized histochemical procedures
handling. • Ex.
o Paraformaldehyde
METHODS OF FIXATION o Osmium tetroxide
- MEANS 2 BASIC MECHANISMS OF FIXATION
• Physical A. ADDITIVE
o Heat Fixation - chemical constituent is taken in & becomes part of
o Microwave Fixation tissue by forming cross-links or molecular complexes and
o Cryopreservation (freeze-drying) giving stability to the protein.
• Chemical
o Immersion fixation - Ex.
o Perfusion fixation • formalin,
o Vapor fixation • mercury,
• osmium tetroxide
-2 BASIC MECHANISM A. NON-ADDITIVE
• Additive Fixatives - fixing agent is NOT incorporated into the tissue
o Formalin
o Mercury - removes the bound water attached to H-bonds of certain
o Osmium tetroxide groups within the protein molecule
• Non-Additive Fixatives • new cross-links are established within and
o Alcoholic fixatives among the protein molecules that stabilize the
intercellular components,
- OTHER TYPES o prevent autolysis and bacterial
• Single Fixative decomposition.
o Dissolved in water or alcohol
• Buffer Solution - Ex.
o Stabilize pH • alcoholic fixatives
• Combination
o defects in one agent can be
compensated for by the addition of BENEFITS OF FIXATION
another 1 Allows thin sectioning of tissue by hardening tissue
▪ Ex. acetic acid = counteract
shrinkage
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Prevents autolysis and inactivates infectious agents

2
(except prion diseases) - As the fixative molecules bind to the tissue, they are
3 Improves cell avidity for special stains eventually depleted.
• Causes poor fixation and staining artefacts
PRACTICAL CONSIDERATION TO OPTIMIZE • Solution: change the solution at intervals to
FIXATION OF TISSUE avoid exhaustion of the fixative.
Specimens should be transferred to fixative quickly
1 - AGITATION will also enhance fixation of the specimen.
(<1hr) after surgery
Tissues should be fixed in a sufficient volume of 2. HYDROGEN ION CONCENTRATION
solution for efficient penetration - best caried out close to neutral pH
2 • ideal ratio = (fixative to specimen) • pH 6-8
o 20:1
o at least 10:1 - HYPOXIA
Anatomical barriers to fixation must be removed or • lowers pH → causes excessive acidity
incised o Acidity favors formation of FORMALIN-
• fascia, HEME PIGMENT
• bone, ▪ Black, polarizable deposits
• feces, in tissue
• thick tissue o BUFFERS = prevent excessive acidity
3 ▪ Phosphate
Large specimens must be _____ to allow penetration ✓ Commercial
• sectioned or inflated with fixative formalin = buffered
o ex. lung w/ phosphate at pH 7
• opened and cleaned ▪ Bicarbonate
o ex. GI tract ▪ Cacodylate
Fixatives diluted and/or contaminated by bodily fluids ▪ Veronal
will be reduced in concentration and must be 3. TEMPERATURE
4 replaced to ensure effectiveness. - TEMP
• Ex. bile, blood, feces • 40 C = regular tissue processing
o Replace or reduced • 0-4 C = electron microscopy & histochemistry
Pinning specimens to corkboard or inserting a • Room temp = mast cells & electron
5 paper or gauze “wick” into tubular structures can microscopy
improve fixation and reduce tissue distortion. o maintain excellent morphological
Sufficient time must be allowed for penetration of detail
fixative
6 - REFRIGERATION slow down decomposition if the
• Rate of penetration vary according to
fixation type tissue needs to be photographed and cannot be fixed
immediately
The size of tissue blocks should be small enough
to allow adequate permeation of fixative (and • Brain cells = deteriorate very quickly.
7
subsequent processing solutions) through the • Bone Marrow = can still undergo mitosis 30
perforations in cassettes. minutes after death
Prolonged fixation may be more difficult to reverse • Nucleic acid = do not react at room temp
8 and may also result in loss of immunohistochemical
antigenicity - Increasing temperature = increased speed of fixation
The tissue hardens upon fixation and remains in • Ex. Use of heat in bacteriology and blood films
whatever shape it was fixed in
9 - An increase in temperature can increase the rate of
• Considered in cases where final orientation
is important. fixation but can also increase the rate of autolysis.
• ↑ temp = ↑ fixation rate = ↑ autolysis
MAIN FACTORS INVOLVED IN FIXATION • Ex. Formalin
1 Volume o Heated up to 60 C for rapid fixation of
2 Hydrogen Ion Concentration very urgent biopsy specimen
3 Temperature ▪ Cons: risk of tissue distortion is
4 Thickness of section increased
5 Osmolality
4. THICKNESS OF SECTION
6 Concentration
- should be small and thin
7 Duration of fixation
• Small
8 Time Interval
1. VOLUME o electron microscopy: 1-2 𝒎𝒎𝟐
- amount of fixative should be 10-20x the volume of o light microscopy: 2 𝒄𝒎𝟐
tissue to be fixed. • Thin
• most common error in histotechnology is o light microscopy = 0.4cm
insufficient ratio of tissue volume to fixative
volume
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- Large solid tissue should be opened or sliced thinly -FIBROUS organ take longer to fix than small or loosely
to improve penetration of fixatives. textured tissue
• Ex. Uterus • Ex. Fibrous = uterus, intestinal tract
• Requires more fixative and longer fixation time • Ex. Loose = biopsies or scrapings

- Fecal matter and stomach contents can inhibit the -time can be cut down by using:
penetration of fixative and damage tissue and therefore • Heat
must be removed before fixation • Vacuum
• Agitation
- Most tissues can be cut and trimmed without prior • Microwave
fixation, except for BRAIN
• soft when unfixed. - FORMALIN carried out for 2-6 hours
• Must be fixed before “grossing” or “sectioning” • May remain in fixative over the weekend
o Suspended in 10% buffered formalin without much adverse affect
for 2-3 weeks. • Washed out after 24 hours

- Penetration of tissue depends upon the diffusability of -PROLONGED FIXATION may cause
each individual fixative, which is a constant. • Shrinkage
• Best • Hardening of tissue
o Formalin • Severely inhibit enzyme activity and
▪ Slow penetration immunological reaction
▪ Solution: open tissue and left o Washing restores enzyme activity
to fix
o Alcohol -Electron microscopy carried out for 3 hours then
• Worst placed in a holding buffer
o Glutaraldehyde 8. TIME INTERVAL
▪ Solution: section tissue thinly - fixed immediately after removal or death to prevent
(2-3mm) autolysis or putrefaction.
• In between (moderate) • Longer blood supply interrupted → poor quality
o Mercurial of tissue.
- Ideally, fixation is done slowly over 1 day or more. - ARTEFACTS = result of drying
• NOT practical • Keep tissue moist with saline
• Alternative
o Time: 4-6 hours - IF fixation is not carried out under optimal conditions/
o Size: 2cm2 and <4mm thick delayed:
• Aldehyde = 2-3mm/hr • More organelles lost
• Solid material (ex. Liver) = <10-15mm • More nuclear shrinkage
5. OSMOLALITY • More artefactual clumping
- recommended = NORMAL PHOSPHATE BUFFERED • Irreversible damage
SALINE (PBS) based fixative:
• Sucrose = added to osmium tetroxide for EFFECTS OF FIXATIVES IN GENERAL
electron microscopy
Reduce the risk of infection during handling and
1
actual processing of tissues.
-results
Harden soft and friable tissues and make the
• Hypertonic = cell shrinkage handling and cutting of sections easier
2
o Best: SLIGHTLYHYPERTONIC = 400-
• Accelerated by alcohol during dehydration
450 mOSm
Make the cells resistant to damage and distortion
• Hypotonic and Isotonic = cell swelling
3 caused by the hypotonic and hypertonic solutions
o Isotonic = 340 mOsm
used during tissue processing
4 Inhibit bacterial decomposition
6. CONCENTRATION
Increase the optical differentiation of cells and
- should be adjusted to the lowest level possible
5 tissue components thereby rendering them more
• Too high = produce artefact similar to that readily visible during examination
caused by excessive heat
May act as mordants or accentuators to promote
• Formaldehyde = 10% solution and hasten staining, or they may inhibit certain
• Glutaraldehyde = 3% solution 6 dyes in favor of another
o 0.25% = ideal for immunoelectron • Formaldehyde = intensifies
microscopy
• Osmium trioxide = inhibits hematoxylin
- buffer cause polymerization of the aldehyde, with
consequent decrease in its effective concentration CHARACTERISITC OF GOOD FIXATIVE
1 Cheap
7. DURATION OF FIXATION
2 Stable
3 Safe to handle
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Kills the cell quickly thus producing minimum • 10% formal saline
4
distortion of cell constituents • 10% neutral buffered formalin
5 Inhibit bacterial decomposition and autolysis • Heidenhain 's Susa
6 Produce minimum shrinkage of tissue • Formal sublimate (formal corrosive)
7 Permit rapid and even penetration of tissues • Zenker 's solution
9
Must harden tissues thereby making the cutting of • Zenker-formal (Kelly 's solution)
sections easier • Bouin's solution
It must be isotonic, causing minimal physical and • Brasil's solution
chemical alteration of the cells and their B. CYTOLOGICAL FIXATIVES
constituents -preserve specific parts and particular microscopic
elements of the cell itself.
There are some hypotonic solutions used in
10
conjunction with hypertonic solution to produce - TYPES
compound which give an optimal effect on tissue • Nuclear Fixatives
structure
• Cytoplasmic Fixatives
• Ex. Hypotonic = glacial acetic acid
• Histochemical Fixatives
• Ex. Hypertonic = picric acid
Make cellular components insoluble to hypotonic A. NUCLEAR FIXATIVES
11 solutions and render them insensitive to -preserve the nuclear structures in particular
subsequent processing. • ex. chromosomes
Must permit the subsequent application of many -primary component: glacial acetic acid
12 staining procedures to facilitate easier and more • due to its affinity for nuclear chromatin.
profitable examination. -pH: < 4.6
-EXAMPLES
TYPES OF FIXATIVES • Flemming's fluid
-choice of the fixative is based on tissue and anticipated • Carnoy's fluid
ancillary tests. • Bouin's fluid
4 MAJOR GROUPS OF FIXATIVES
• Newcomer's fluid
GROUP EXAMPLE ACTION
• Heidenhain's Susa
- formaldehyde, - can react with nucleic acids
Aldehydes
- glutaraldehyde
cross-linking • Mercury
- osmium tetroxide,
Oxidizing proteins • Chromium salts
- potassium
Agents - Mercuric chloride react with viruses, and causes the
permanganate
loss of their infective power
- methyl alcohol, protein-
Alcohol-Based
- ethyl alcohol, denaturing B. CYTOPLASMIC FIXATIVES
Fixatives
- acetic acid agents - preserve cytoplasmic structures in particular.
insoluble - never contain glacial acetic acid which destroys
Metallic - mercuric chloride
metallic mitochondria and Golgi bodies of the cytoplasm.
Fixatives - picric acid
precipitates -pH: >4.6.
ACCORDING TO COMPOSITION • Flemming's fluid without acetic acid
A. SIMPLE Fixatives B. COMPOUND Fixatives • Kelly's fluid
-are made up of only one - are those that are made • Formalin with "post-chroming"
component substance. up of two or more
• Regaud 's fluid (Muller 's fluid)
1. Aldehydes fixatives which have been
• Orth 's fluid
• Formaldehyde added together to obtain
- RNA = uses precipitant fixative on a frozen tissue
• Glutaraldehyde the optimal combined
• Ethanol
2. Metallic Fixatives effect of their individual
• Acetone
• Mercuric chloride actions upon the cells and
• Chromate tissue constituents.
C. HISTOCHEMICAL FIXATIVES
fixatives - preserve the chemical constituents of cells and
3. Picric acid tissues.
4. Acetic acid a) Formal Saline 10%
5. Acetone b) Absolute Ethyl Alcohol
6. Alcohol c) Acetone
7. Osmium Tetroxide d) Newcomer's Fluid
ACCORDING TO ACTION
A. MICROANATOMICAL FIXATIVES SECONDARY FIXATION
-are those that permit the general microscopic study of - placing an already fixed tissue in a second fixative in
tissue structures without altering the structural pattern order:
and normal intercellular relationship of the tissues in • To facilitate and improve the demonstration of
question.
particular substances.
-EXAMPLE • To make special staining techniques possible

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o secondary fixative acting as a mordant o Small tissue biopsies may be

• To ensure further and complete hardening and placed in a petri dish with
preservation of tissues. moistened filter paper
Distortion, rough handling or other mechanical
- done before dehydration and on deparaffinized sections 6 damage to the tissue will cause permanent
before staining, morphological damages
• Primary fixative: 10% formalin or 10% formol If placed in normal saline solution (NSS) during the
saline. 7 operation, autolysis may occur before fixation is
carried on.
-10% buffered neutral formalin may require secondary Tissues should NOT be >5 mm thick with minimum
fixation with Zenker’s solution prior to staining 8 squeezing and handling
• Zenker = acts as mordant • Except: lung edema = 1-2cm
• Stains Purulent material, exudates or transudates should
o Masson's trichrome = connective 9 be marked and kept for possible cultures,
tissue smears, and other bacteriologic examination
o Mallory's aniline blue stain = Amount of fixative approx. 10-20x the volume of the
collagen, 10 tissue specimen
o Phosphotungstic acid-hematoxylin • Osmium tetroxide = 5-10x of tissue volume
(PTAH) stain = striated muscle For prolonged fixation volume of fixing fluid should
POST-CHROMATIZATION 11 not be less than 50 times that of the tissue
- primarily fixed tissue is placed in aqueous solution of • e.g. museum preparation
2.5-3% potassium dichromate for 24 hours Contamination of fixed tissue with precipitates
• act as mordant for better staining effects should be avoided. In most instances, fixed tissues
• aid in cytologic preservation of tissues. 12 must be washed thoroughly to remove salts
WASHING OUT and/or pigments before staining.
- removing excess fixative from the tissue after fixation • Ex. Osmium tetroxide
in order to improve staining and remove artefacts from the The required period for fixation should not be
tissues. 13 exceeded since this may cause excessive hardening
Tap water is used to remove: or brittleness of tissues.
• excess chromates from tissues fixed in Hollow organs should be packed with cotton
o Kelly's, soaked in fixative or completely opened before
14
1 o Zenker's, being immersed in adequate fixing solution
o Flemming's solutions • (e.g. stomach, intestines)
• Excess formalin Air-filled lungs may float on fixative. To avoid this,
• Excess osmic acid 15 the organ may be covered with several layers of
50-70% alcohol is used to wash out excess amount gauze to maintain it under surface.
2 Human brains may be suspended by a cord tied
of picric acid (Bouin's solution).
Alcoholic iodine is used to remove excessive under the Circle of Willis to prevent flattening.
3
mercuric fixatives. • require long fixation, usually 2 weeks prior
16 to sectioning
CHARACTERISITC OF GOOD FIXATIVE • Intravascular perfusion (washing out of
1 Must must be properly labeled and identified. blood with Ringer's lactate) may lead to
Surgical specimens should be fixed ASAP after artifact formation with loss of blood content
removal, or refrigerated if fixation is to be delayed, Eyes should not be dissected before fixed since
2 this may lead to immediate tissue collapse and
to prevent drying of surface layers and ultimate
tissue distortion wrinkling due to the escape of vitreous humor.
17
If fixation is NOT immediately possible, refrigerate • Not easily penetrated due to tough sclera
but do NOT freeze. • Formal-alcohol = injected before
• Slow freezing of unfixed tissues near 0°C = immersing
formation of ice crystals Frozen sections may lead to formation of ice
3 18
• Repeated freezing and thawing = crystal artifacts
o destroy cellular organelles, To avoid rigor contraction and staining of artifacts
o release enzyme, • Stretch fresh biopsy by sutures on each
o diffuse soluble cell components 19 end
Consider any fresh or incompletely-fixed tissue as • Laid flat in moist filter paper before
4 potentially infectious to you and other workers in suspending in fixative
your lab Water should not be used for glycogen-containing
20
Drying should be avoided to prevent shrinkage and tissues because glycogen is soluble in water.
distortion of tissue with loss of cellular detail. Tissue may be minced that may be divided into
5 • Causes permanent damage and mask any 21 small fragments and transferred to the vial of a
pathological change fixative by means of a tooth pick
• Small endoscopic specimens = susceptible Hard tissues may be washed out in running water
22 overnight and immersed in 4% aqueous phenol for
1-3 days (Lendrum’s method)

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• Ex. Cervix, uterine, fibroids, hyperkeratotic Tissue blocks are

Prolonged fixation
skin brittle and hard
Presence of mucus prevents complete FIXATION ARTIFACTS
penetration of fixative; - FORMALIN PIGMENT is a well-known artifact that may
23
• Excess mucus may be washed away with be produced under acid conditions.
normal saline solution. • Due to nonbuffered formaldehyde
Fatty tissues should be cut in thin sections and • eliminated or reduced by fixation in PHENOL-
24
fixed longer. FORMALIN
Tissues containing large amount of blood (e.g. o and neutral buffered formalin
25 blood vessels and spleen) should be flushed out
with saline before fixing - "CRUSH ARTIFACT"
The position or shape that is desired for sectioning • surgical specimens w/ intense eosinophilic
should be maintained before fixation. staining at the center of the tissue in H&E
26
• Ex. Intestine = stretched out on a paper and stained sections
placed in container → fixate o Ex. Liver biopsies
The fixative should penetrate from all sides • Due to partial coagulation of partially fixed
• Always place specimens into containers protein by wax
27
that already contain fixative • Incomplete wax impregnation
o Prevents adhesion
Where possible, hollow organs or specimens with LIPID FIXATION
28 natural cavities should be opened to allow − conventional histopathological technique, lipids are
immediate access to the fixative. removed during preparation of tissues
Gentle agitation (swirling) of the specimen during − Cryostat or frozen sections used for demonstrating
29 its first few minutes in fixative will facilitate lipid in tissues then lipid stain.
penetration − Fixatives containing MERCURIC CHLORIDE AND
Fixatives should be carefully made from reagents of POTASSIUM DICHROMATE → preservation of
suitable quality, and fresh if so specified lipids in cryostat sections.
30 • Poor quality reagents = poor quality fixation − ALDEHYDES→ phospholipids (contain amino
• Helly’s fluid = prepared from stock solution groups)
o Unstable ▪ formaldehyde → react with unsaturated fatty
If a specimen is received in fixative of dubious acids during histological fixation→less lipid can
31
quality, it must be replaced with fresh fixative. be demonstrated histochemically in tissue that
Fixatives should be used only once has been stored in formaldehyde for a long time.
32 • Specimens shed cells and tissue fragments − BAKER'S FORMAL-CALCIUM → preserve
into the fixative solution → contamination phospholipids.
Avoid metal lids − post-fixing in IMIDAZOLE OSMIUM TETROXIDE→
33
• Mercury salts = corrosive improved ultrastructural of lipids
Some fixatives require that specimens be washed in − DIGITONIN → cholesterol ultrastructural
water prior to processing demonstration
34
• Zenker’s
• Helly’s CARBOHYDRATE FIXATION
All fixatives are toxic and irritant. Anyone using − Carbohydrates → hydrophilic
35
fixatives should be aware of their potential hazards. − Alcoholic fixatives (ROSSMAN'S FLUID OR COLD
ABSOLUTE ALCOHOL) → glycogen fixation
DIFFICULTIES CAUSED BY IMPROPER FIXATION: ▪ glycogen storage disease
CAUSE ▪ coated with celloidin: better retention of glycogen
- Failure to fix immediately ▪ 60-80% losses of glycogen can be high in
Failure to arrest early aqueous solution
(dry before fixing)
autolysis of cells − Alcoholic formaldehyde → better fixative in human
- Insufficient fixative
Removal of skin compared with neutral buffered formaldehyde.
substances soluble Wrong choice of fixative
in fixing agent PROTEIN FIXATION
Presence of artefact - most commonly used fixatives for amino acid
pigments on tissue Incomplete washing of fixative histochemistry
sections • Neutral Buffered Formal Saline
Tissues are soft and • Formaldehyde Vapor
feather-like in Incomplete fixation ELECTRON MICROSCOPY
consistency − whole procedure performed at 4°C
Loss or inactivation a) osmium tetroxide,
of enzymes needed Wrong choice of fixative b) glutaraldehyde and
for study c) paraformaldehyde,
Shrinkage and ENZYME HISTOCHEMISTRY
Overfixation
swelling of cells and a) 4% formaldehyde or
tissue structure b) formal-saline overnight, or
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c) fresh frozen cryostat sections − Cryosection morphology: often inferior to paraffin

▪ may be fixed in acetone or formaldehyde sections
▪ and washed in distilled water prior to enzyme
staining. Formalin-fixed-paraffin
IMMUNOFLUORESCENCE − embedded tissue sections typically 'auto fluoresce'
− pathology → various antibodies when viewed under a fluorescent microscope
− more sensitive cases→ tissue from cryostat section − immunofluorescent (IF) staining → best done on
→ fixation limited to a few seconds in absolute frozen sections.
methanol or acetone. − Background auto fluorescence, to a greater or lesser
▪ Insufficient fixation →unfixed tissues→ damaged degree, can be suppressed → post-staining Sudan
by dehydrating fixative effect of ethanol during Black treatment
processing →loss of immunohistochemical
antigenicity and poorer microtomy.
▪ Antigen-retrieval techniques are required for Fixation
successful immunohistochemistry. -Fix cells in -20°C ACETONE for 5-
IMMUNOHISTOCHEMISTRY 10 minutes.
Acetone
− Some antibodies cannot recognize antigens if they -NO PERMEABILIZATION step
are cross-linked in a certain manner. needed following acetone fixation
▪ Antigen retrieval may be used to uncover -Fix cells in -20°C METHANOL for
antigens after fixation. 5-10 minutes.
Methanol
− Immunocytochemistry→ sample preparation →fixing -Permeabilization step is needed
the target cells to the slide. following methanol fixation
− Perfect fixation Fix cells in cooled 95% ethanol,
▪ immobilize the antigens, Ethanol 5% glacial acetic acid for 5-10
▪ while retaining cellular and subcellular minutes
architecture and -Fix in cooled methanol, 10
▪ permitting unhindered access of antibodies to all minutes at –20 °C.
Methanol-Acetone
cells and subcellular compartments. -Remove excess methanol.
Fixation
− Fixation methods → 2 classes: organic solvents and -Permeabilize with cooled acetone
cross-linking reagents. for 1 minute at –20 °C
a) Organic solvents -1:1 methanol and acetone
Methanol-
▪ alcohols & acetone ACETONE MIX
mixture.
▪ remove lipids and dehydrate the cells, while Fixation
-Make the mixture fresh and fix
precipitating the proteins on the cellular cells at -20 C for 5-10 minutes
architecture. Methanol- -1:1 methanol and ethanol mixture.
ETHANOL Mix -Make the mixture fresh and fix
b) Cross-linking reagents Fixation cells at -20 C for 5-10 minutes
▪ paraformaldehyde -Fix 5-10 minutes.
▪ form intermolecular bridges→network of linked 10% neutral -Rinse briefly with PBS.
antigens. buffered formalin -Permeabilize with 0.5% Triton X-
▪ Cross-linkers preserve cell structure better than 100 for 10 minutes
organic solvents, but may reduce the antigenicity -Fix 10-20 minutes.
3-4%
of some cell components, and require the -Rinse briefly with PBS.
Paraformaldehyde-
addition of a permeabilization step, to allow -Permeabilize with 0.5% Triton X-
TRITON
access of the antibody to the specimen. 100 for 10 minutes
− Fixation w/ both methods →may denature protein -Fix 10-20 minutes.
antigens → antibodies prepared against denatured 4% -Rinse briefly with PBS.
proteins may be more useful for cell staining. Paraformaldehyde- -Permeabilize with cooled
− Alcohol based fixation → dehydrates cells/tissues, METHANOL methanol for 5-10 minutes at –20
causing proteins to denature and precipitate in situ. °C
− Paraformaldehyde causes covalent cross-links
between molecules → insoluble meshwork. PRACTICAL CONSIDERATION
− Fixation method, time, and temperature are variables 1. Do not reuse or save diluted antibodies.
that need to be considered when developing ▪ antibodies adhere to the walls of the container
immunohistochemistry protocols and this effectively reduces the titer to zero.
2. Freeze / thaw cycles will destroy antibodies.
Cryopreservation 3. 'Dirty' (high background) polyclonal antibodies can
− avoid or reduce the ‘auto fluorescence’ (ex. high sometimes be 'cleaned up' by storing the antibody
background) that is inherent to formalin fixed paraffin over pieces of fixed tissue.
→ immunofluorescent microscopy. ▪ Irrelevant antibodies in the serum may be
− It promotes the preservation of delicate enzymes that adsorbed onto the tissue, effectively raising the
are easily destroyed by fixation. titer of the specific antibodies.
− Lipid stains (ex. Oil Red O) require frozen sections ▪ incubate the antibody on a tissue section and
since the ethanols used in tissue processing will then reuse the antibody on a second section.
extract the lipids.
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ANTIGEN RETRIEVAL FOR IMMUNOSTAINING − specimen → buffered formalin or other fixative →later
− reactions of fixation are reversible (formaldehyde) stage →microwaved to assist the fixative action of the
− number of methods → restore antigenicity → not fixing agent
used when working with cryo-preserved sections − microwaving while tissue is in fixative→ some hazard
from toxic fumes produced, or tissue may be
− Microwave antigen retrieval is one of several so- transferred back to saline or buffer for the microwave
called ‘HIER’ (heat induced epitope retrieval) step.
methods. − fixative solution → within the tissue → microwave-
− Others include steaming, pressure cookers and assisted fixation
autoclaves. − more commonly used than primary microwave
▪ involves boiling the slides in a pH 6 citric acid fixation.
buffer for 10 minutes, followed by an additional − Proprietary fixatives of relatively low toxicity
15 minutes in the hot buffer. containing glyoxal, have been developed for use in
− changing the pH to 3 (in citric acid buffer) or to 8-9 (in microwave-assisted fixation.
a Tris EDTA buffer) are successful. − 2 mm thick slices initially fixed in formalin prior to
microwave treatment
− Partial digestion of tissue sections by enzymatic
method with one (or several) enzymes can − rate microwave energy will generate heat during
sometimes be effective. tissue fixation depends on a number of factors
▪ power setting and power output of the oven,
EFFECT OF HEAT DURING FIXATION ▪ volume and nature of the holding solution, t
− fixative temperature → raised or lowered → rate of ▪ composition, shape and number of containers
diffusion into the specimen is affected (including cassettes),
− Increasing temperature →accelerates the process of ▪ agitation or movement of the containers,
fixation. ▪ number, volume and dimensions of the
− prolonged excessive heat → damage the cells and specimens being fixed.
cause substantial shrinkage and hardening of the − After microwaving → immediately be sliced to 2 mm
specimen. → 70% ethanol.
− most laboratories → primary fixation → ambient − many variables involved in microwave fixation→ fully
temperature (37°C to 45°C) standardized (consistent specimen dimensions),
− hot fixative solutions → fix larger specimens (>3mm microwave oven is properly calibrated, and staff fully
thick) understands the factors that will influence its outcome
▪ outside of specimen fixes rapidly but slow Advantages
penetration in center block → poorly fixed or not 1. chief advantage: tissue is heated right through the
fixed at all. block in a very short time → rapid study of cellular
▪ blocks → exaggerated “zonal” fixation effect with processes
different morphological and staining 2. It is a non-chemical technique that is useful in
characteristics on the outside as compared to the preserving neurochemical substances in brain
inside of the specimen. (acetylcholine).
3. rapid fixation of routine surgical specimens.
4. reduces the time taken for immuno-histochemistry
MICROWAVE FIXATION
and in-situ hybridization.
− commercial microwave ovens → easy access to
5. Microwave treated tissue (at 50C), post-fixed in
controlled heating → overcoming the previous
osmium tetroxide also gives satisfactory results for
problems of erratic heating (by direct flaming)
electron microscopy.
− Microwave fixation allows light microscopic
Disadvantages
techniques used in routine histopathology to be
1. Microwaves generated by commercial ovens only
performed adequately.
penetrate tissue to a thickness of 10-15 mm.
− standard stains, special stains, histochemistry for
2. There is no significant cross-linking of protein
mucous substances, some enzyme histochemistry,
molecules, and subsequent chemical fixation may be
and immuno-cytochemical techniques.
needed.
− Heat→major factor → effects of microwaves during 3. Viable spores and other pathogens may remain in
tissue fixation. tissues processed with alcohol-based fixatives or
− increasing diffusion rates & molecular kinetics and microwaving alone.
speed up chemical reactions. FIXATIVE
− temperature > 60°C → primary microwave fixation; − M Phosphate buffered,
lower temperatures → microwave-assisted fixation.
− 40% Formaldehyde (10% formalin buffered to pH 7.3)
▪ Expose tissue to formaldehyde, followed by 1.5
2 ways microwave technology → tissue fixation
minutes of microwave
1. MICROWAVE FIXATION OR MICROWAVE
PROCEDURE
STABILIZATION:
1. Fix tissue (no more than 5 mm) in formalin solution
− fresh tissue→ saline or other isotonic solution →
for 4 hours.
primary fixation
2. Soak blocks in water at RT for 1 minute in 100 ml of
− No chemical fixative is used at this stage.
formalin.
2. MICROWAVE-ASSISTED FIXATION:
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HISTOPATH LEC MIDTERMS
LECTURE / TOPIC 6 TRANSCRIBED BY: IVAJ
DO NOT REDISTRIBUTE W/O PERMISSION!

3. Place in microwave, 450 watts, at 55°C for 1.5 to 4

minutes.
4. Remove blocks and slice tissue to 2 mm thick.
5. Place directly in alcohol.

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