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histopathology notes
fixation
 it stops the activity of enzymes
 DEFINE FIXATION  it prevents the breakdown of cellular
 GOALS OF FIXATION
components (autolysis – release of enzymes
 FACTS ABOUT TISSUE PROCESSING
inside the cell that will lead to the breakdown
 FACTORS AFFECTING FIXATION
of several cellular components)
 CONSIDERATIONS IN FIXING TISSUES
 it allows retention of certain tissue components
 METHODS OF FIXATION
 FIXATION MECHANISMS FACTORS AFFECTING FIXATION
 TYPES OF FIXATION
 SECONDARY FIXATION  volume (amount of fixative to be placed in
 WASHING OUT contrast to the amount of fixative); general
 BIOCHEMICAL FIXATION rule: volume of fixative must be 20x greater
 FIXATION FOR EM, ENZYME HC, IF AND than the volume of the tissue; 1:20
IHC range: 10-25x volume of fixative
 CHEMICAL FIXATION  hydrogen ion concentration (pH) (almost
 DE-ZENKERIZATION neutral pH; 6-8 slightly alkaline and acidic)
 MICHEL'S SOLUTION  temperature (cool; retard , warm; enhance the
diffusion of fixative)(high temp above 45C;
FIXATION
maceration of tissue, some procedures may
 process of preserving tissues require the tissue in a ref temp for e.g. electron
 prevents decay microscopy; temp must be 4C or less, frozen;
 retains the state of the tissue as to how it was freezing temp around -10C - -5C made
removed from the body possible by optimal cutting temperature)
 inactivates enzymes inside the cell,  thickness of section (thicker less fixative will
preventing autolysis penetrate tissue; thinner the better fixative will
 bacterial contaminants that may have penetrate)
attached to the tissue will also be killed thus  osmolality (kept at a slightly hypertonic
preventing the process of putrefaction osmolality)
 stabilizes cellular structures by making  concentration (not all concentrated fixative
macromolecules resistant to water will provide best results, all concentrated
 enzymes are proteins fixatives will contribute brittleness to tissues so
 fixatives denature proteins u must dilute fixatives to the point that
brittleness is eliminated and will provide
GOALS OF FIXATION
enough water to contribute to volume of tissue
1. preserves the chemical and morphological but to a point that it will swell)
integrity of the cell; ‘lifelike”  duration of fixation (the duration of fixing a
2. harden and protect the tissue [tissue will be tissue will depend on how thick or thin the
subject to compressing forces when you cut it tissue and the type of fixative used) (metallic
via microtomy] fixative; hours)
 time interval (must be submerged at least 1
FACTS ABOUT TISSUE PROCESSING hour after removed from the body to avoid
 fixation makes cell slightly swell (fixatives that cellular death of tissue)
contain great amount of water like; 10% CONSIDERATIONS ON FIXING TISSUES
formalin solution)
 tissue loses 20% - 30% of their total volume  tissues must be fixed within 1 hour
 it affects the degree of staining (retard or  tissues to fixative ratio must be at least 1:10 or
enhance) in general 1:20
 anatomical barriers must be incised or inflated
1
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histopathology notes

 large specimens must be sectioned or inflated tissues, can cause confusion to the reading so
 pin tissues on a corkboard or insert a wick into it must be removed before staining)
tubular structures to maintain shape
 duration and rate of penetration must be
According to COMPOSITION
considered
 prolonged fixation can result in the loss of A. Simple Fixatives (one component)
IHC/Immunohistochemistry antigenicity (detect
antigens on the tissues)(ag ab interaction)  Contains one component
 tissues harden during fixation (epitopes will be 1. Aldehydes
lost) 2. Metallic Fixatives
3. Picric Acid
METHODS OF FIXATION 4. Acetic Acid
5. Acetone
1.PHYSICAL
6. Alcohol
 Heating
7. Osmium Tetroxide
 Microwaving
 Cryo-preservation (frozen tissues) B. Compound Fixatives (one, two or all are
2.CHEMICAL FIXATION combined)
 Immersion fixation (most common)
 Perfusion fixation (fixative is ingested during  Combination of two or more fixatives.
embalming process)  Combines effects of the individual action of
 Vapor-fix (heat will produce vapor of fixative fixatives.
thus producing vapors that will bind to the II. According to ACTION
tissue)
A. Microanatomical Fixatives (preserve structure
FIXATION MECHANISMS of entire cell)
 Additive fixation – fixative is incorporated  Permits microscopic study
inside the cells and it forms cross links or  Doesn't distort the structural patterns of the
complexes, providing stability cells
 Non-additive fixation – alters the tissue
composition and removes water from tissues B. Cytological Fixatives (preserve a certain part of
by competing with the H-bonds formed by the cell)
water with molecules
 Preserves SPECIFIC parts
TYPES OF FIXATION  NUCLEAR (≤ pH 4.6) - preserve contents of
nucleus (acidic)
FOUR MAJOR GROUPS:  CYTOPLASMIC (> pH 4.6) - preserve
 Aldehydes - Cross-links proteins (denatured cytoplasmic contents (somewhat basic ph)
in the process but will deposit on the  HISTOCHEMICAL ( preserve biochemical
cytoskeleton to provide turbidity) molecules inside tissue e.g lipids fats proteins
 Oxidizing Agents - Cross-links proteins and nucleic acids)
(denatured in the process but will deposit on SECONDARY FIXATION
the cytoskeleton to provide turbidity)
 Alcohol-based Fixatives - Protein-denaturing  Fixing an ALREADY PRESERVED tissue in a
agents (denatured and precipitated outside the different fixative.
cell)  Demonstration of particular substances. (lipids
 Metallic Fixatives - Forms insoluble metallic and other types)
precipitates (enhance the staining qualities of  Makes special staining possible (MORDANT).
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histopathology notes

 Ensures further and complete hardening and ELECTRON MICROSCOPY

preservation of tissues.
 Done at 4°C
 Done before dehydration and on
 Fixatives containing glutaraldehyde
deparaffinized tissues before staining.
 Post-Chromatization using potassium ENZYME HISTOCHEMISTRY
dichromate (tissues have an enhanced
staining quality  Maximal preservation of enzyme activity to
where it originates, while also preserving
WASHING OUT structural integrity.
 Overnight fixation using 4% formaldehyde or
 Process of removing excess fixative.
formal-saline
 Improves staining; removes artifacts.
 Fixed with acetone or formaldehyde for frozen
Solutions used: sections.
 Tap Water (found in faucet, used to rinse out IMMUNOFLUORESCENCE and
excess fixative) IMMUNOHISTOCHEMISTRY
 50% - 70% Alcohol (necessary to rinse out
 Epitopes of antigens must be preserved for
fixatives- fixatives fixed in citric fixatives)
antibodies to bind on antigens to be detected.
 Alcoholic lodine (removing deposits formed
 Antigen retrieval can be done to bring back
by metallic fixatives especially mercury
antigenicity using Heat-induced Epitope
fixatives)
Retrieval (HIER).
MICROWAVE FIXATION
BIOCHEMICAL FIXATION
 Employing the advantage of heat to fix tissue,
LIPID FIXATION
microwaves aid in the fixation process by
 Hydrophobic either allowing heat to permeate and fix the
 Must be fixed with a solution that mustn't tissue itself (microwave fixation) or heat using
affect lipid composition. microwave assists the permeation of a
 Frozen tissues stained with a general lipid chemical fixative (microwave-assisted fixation).
stain.
ADVANTAGES:
 Use fixatives containing HgCI2 or potassium
dichromate.  Allows examination of rapid cellular processes.
 Phospholipids are fixed with aldehydes  Non-chemical; allows preservation of
(Baker's formol-citrate). neurochemical substances.
 Digitonin for cholesterol demonstration.  Rapid fixation of tissues of routine surgical
specimens.
CARBOHYDRATE FIXATION
 Significantly reduces time for IHC and in-situ
 Hydrophilic hybridization.
 DO NOT USE FIXATIVES WITH HIGH  Allows the satisfactory production of tissues for
WATER CONTENT EM.
 Alcoholic fixatives (Rossman's fluid)
DISADVANTAGES:
PROTEIN FIXATION
 May only penetrate tissues with 10-15 mm
 For amino acid histochemistry, thickness.
 NB Formol-Saline or Formaldehyde vapor.  No significant cross-linking of proteins.
 Spores and other pathogens may remain in the
FIXATION FOR EM, ENZYME HC, IF and IHC
tissues.
3

histopathology notes
CHEMICAL FIXATION
 Use of chemical reagents to preserve tissues.
 Chemical fixatives have their own different NOTABLE ALCOHOLIC FIXATIVES
actions. (- Cross-links with proteins, providing
additional rigidity to the cells. - Denatures
proteins.)
FORMALDEHYDE and FORMALIN
 Sold as 35% - 40% w/v formaldehyde solution.
 Diluted to a 10% formalin solution in
phosphate-buffered saline to meet adequate
fixative properties.
 Cheap, easy to prepare, penetrates tissues
well, and can be stored for a long time.
PARAFORMALDEHYDE
 White powder; formaldehyde polymers.
 Allows paraffin embedding and sectioning, and
IHC.
 Effects are reversible by excess water.
 Avoids pigmentation produced by formalin.
 Good tissue penetration. Long term storage.
GLUTARALDEHYDE
 2%, 2.5% or 4% METALLIC FIXATIVES
 Standard fixative for EM
 2% - EM
 2.5% - Small tissue biopsies and needle
biopsies
 4% - for larger tissues less than 4 mm thick
 Pleasant and less irritating on the nose.
KARNOVSKY'S FIXATIVE MERCURIC CHLORIDE
 4% Paraformaldehyde-1% Glutaraldehyde in
0.1 M Phosphate Buffer
 Suitable to use when tissues are to be
embedded in resin.
 Suitable fixative for electron microscopy.
ALCOHOLIC FIXATIVES
 Denatures proteins
 Not routinely used for tissues
 Rarely used alone for fixing blocks, unless
studying nucleic acid.
 Good for cytologic smear fixation OTHER NOTABLE METALLIC FIXATIVES
 Used as 70% to 100% concentration.
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 Mixed with acetic acid, water, and/or
chloroform to reduce distortions.
 Methanol - PBS, dry and wet smears, and
BMA smears
 95% Isopropanol - Touch preps
 Ethanol (70 - 100%) used as a fixative and a
dehydrating agent
 Carnoy's Fluid (Absolute alcohol in
Chloroform) - preserves Niss! bodies,
cytoplasmic granues, nucleoproteins, nucleic
acids; for fixing brain tissues for rabies
 Alcoholic Formalin - fixation or post-fixation
of large fatty specimens
 Clarke's Solution - can be used on frozen
sections
 Formol-Acetic Alcohol - Sometimes used for
fixing tissues for diagnostic cryostat
 Gendre's Fluid - enhances
immunoperoxidase studies; for glycogen and
micro-incineration
 Newcomer's Fluid - for fixing
mucopolysaccharides and nuclear proteins
 Actions of these fixatives are still not
understood.
 Allows a good to excellent staining quality.
 Poor penetration; produces shrinkage.
 Ideal for hematopoietic and reticuloendothelial
tissue processing
 Most common metallic fixative
 Used as a 5-7% aqueous solution
 Works by additive and coagulative means.
 Gives excellent nuclear detail.
 Allows excellent trichrome staining.
 Poor penetration and allows significant cell
shrinkage.
 Routine fixative of choice for tissue
photography.
 Mercury deposits may form.

histopathology notes
 Zenker's Solution - recommended for
congested specimens, and for trichrome
staining and PTAH staining
 Zenker-Formol (Helly's) Solution - excellent
fixative for bone marrow, liver (to demonstrate
extramedullary hematopoiesis), spleen,
pituitary glands, and cardiac muscle.
 Lillie's B-5 Fixative - for fixing hematopoietic
BM biopsies, and lymphoid tissues.
 Heidenhain's Susa - for tumor biopsies of the
skin; permits easier sectioning of large blocks
of fibrous connective tissue
OXIDIZING AGENTS
 Permanganates, Dichromates and Chromic
acid, Osmium Tetroxide
 Reacts with various side chains of proteins and
other biomolecules, forming crosslinks,
stabilizing tissue structure.
 May extensively denature despite preserving
fine structures.
 Mainly used as a secondary fixative.
OSMIUM TETROXIDE; OSMIC
 Causes complete denaturation of proteins
 Traditionally used in EM (fixative & stain)
 Good fixative and stain for lipids.
 Preserves cytoplasmic structures.
 Penetrates at a slower rate vs. metallic
fixatives.
 May give off a black stain
 Extremely volatile; prolonged exposure to
vapors may cause conjunctivitis.
OSMIUM-BASED FIXATIVES
 Flemming's Fluid - most common; permanent
fixative for fat
 Flemming's without acetic acid - 
recommended for demonstrating mitochondria
CHROMATE FIXATIVES
 Chromic acid (1-2%) - good in preserving
mitochondria at pH 4.5- 5.2
 Regaud's Fluid - recommended for
demonstrating mitochondria, mitotic figures,
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BSMLS3
Golgi bodies, RBC and colloid-containing
tissues
 Orth's Fluid - recommended for studying early
degenerative processes and tissue necrosis;
also advisable for demonstrating rickettsiae
and other bacteria
PICRATE FIXATIVES
 Performs almost as well as mercuric fixative;
doesn't harden as much.
 Reacts with histones and basic proteins;
precipitates all proteins.
 Good fixative for connective tissues, preserves
glycogen well, extracts lipids.
 May impart a yellow pigment. -Removed using
lithium carbonate or wash in alcohol in
changes of 50-70%
a. PICRATE FIXATIVES
 Bouin's Fluid - recommended for fixing
embryos and pituitary biopsies, also glycogen
 Hollande's Solution - recommended for
gastro-intestinal tract specimen and endocrine
tissues; has some decalcifying properties
 Gendre's Fluid - highly recommended for
glycogen and carbohydrate preservation;
produces minimal distortion of micro-
anatomical structures.
 Brasil's Alcoholic Picroformol Fixative -
better and less mess) than Bouin's; excellent
for preserving glycogen.
GLACIAL ACETIC ACID
 Anhydrous
 Freezes at 16°C
 Enables cell swelling
 Precipitates and fixes nucleoproteins
Compounded with other fixatives to counteract
the swelling effect.
LEAD FIXATIVES
 Recommended for fixing acid
mucopolysaccharides, mucin.
 May produce lead carbonate upon standing.
TRICHLOROACETIC ACID (TCA)
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histopathology notes

 Fixative and decalcifying agent

 Precipitates proteins and nucleic acids.
 Used only on small pieces; penetrates poorly.
ACETONE
 Not recommended as a morphological fixative
 Used in fixing cryostat sections and for tissues
requiring enzyme preservation.
 Used to fix tissues at cold temperatures (0-
4°C)
 Recommended for fixing brain tissues to
diagnose rabies
 Preserves glycogen poorly; dissolves fats.
DE-ZENKERIZATION
 Removal of mercury deposits from tissues
fixed with mercury or mercury-based fixatives.
 Lugol's lodine or an alcoholic iodine solution
complexes with precipitates.
 Sodium thiosulfate (5%) solution is used to
rinse out the complexes formed by mercury
and iodine.
MICHEL'S SOLUTION
- transport to reference lab
 Not a fixative
 Stable medium for transporting fresh, unfixed
tissues that will undergo frozen sectioning or IF
studies.
 Specimens can be kept in the solution for 5
days until it can be delivered to a reference
laboratory.
 Not suitable for keeping tissues for FISH.
 Tissues in such solution must be kept at an
ambient temperature of 4-22°C.