Fixation of Tissues

Major Objective of Fixation

Maintain clear and consistent morphological features.

Good fixation of kidney specimen Bad fixation of kidney specimen

causing crenated cells.
Source: http://classes.midlandstech.edu/carterp/Courses/bio110/chap03/chap03.htm
 When fixing keep in mind:
Stained sections must:

 Maintain the original microscopic relationships among cells,

cellular components, and extracellular material.
 Have little, if any, disruption of the organization of the tissue.
 Preserve the tissue’s local chemical composition.
 Minimize loss of cellular components and macromolecular
structures.
 An Ideal Fixative
 Support high quality and consistent staining with H&E.
 Allow for tissues to be stored for at least a decade.
 Prevent short and long term destruction by intrinsic and extrinsic
factors.
 Permit recovery of macromolecules (proteins, RNA, DNA)
 Be safe – good toxicological and flammability profiles.
 Can be used for a wide variety of tissues
 Provide good penetration for large specimens.
 Have long shelf life, be readily disposable or recyclable and be cost
effective.
Fixation problems (most common)

 Molecular loss from fixed tissues

 Swelling/shrinking
 Inconsistent histochemical/immunohistochemical staining
 Inability to perform accurate biochemical analyses
 Loss in structure of cellular organelles
How do fixatives work?
 Physical methods (uncommon)
• Heating
• Microwaving
• Freeze-drying

 Chemical methods
• Coagulant fixatives
• Cross-linking fixatives
• Compound fixatives
 Heat Fixation
 Simplest form of fixation.

 Acts by precipitating proteins and rendering them less

soluble in water (think about eggs).

 Rarely used on its own. Used to accelerate other forms of

fixation.
 Microwave Fixation

 Speeds fixation (reduces fixation time from 12 hours

down to 20 minutes)

 Disadvantage: produces dangerous vapors that can pose

safety and health problems.
 Freeze Drying

 Very useful in research-based labs for the study of soluble

materials and small molecules.

 Tissue is immersed in liquid nitrogen followed by the

removal of water under vacuum at -40°C.
 Chemical Fixation

Three major categories:

 Coagulant

 Cross-linking (covalent additive fixatives)

 Compound
 Coagulant Fixatives
 Can be organic and non-organic solutions.

 Coagulate proteins, making them insoluble – when lipoproteins and

fibrous proteins (collagen) are coagulated, tissue structure is highly
maintained.

Not useful for ultrastructural analysis due to:

 Poor preservation of mitochondria and secretory granules

 Cytoplasmic flocculation
 Types of Coagulant Fixatives

 Dehydrant coagulants (alcohols such as ethanol and

methanol, acetone).

 Acidic coagulants (picric acid, acetic acid, tricholoracetic

acid).
 Dehydrant Coagulant Fixatives
 Remove and replace free water from tissue (destabilize hydrogen
bonding).

 Results in the disruption of the tertiary structure of proteins

(denaturation) and subsequent coagulation.

 Rate of reversal back to a soluble ordered state is impossible even if

placed in an aqueous environment again.
 Acidic Coagulant Fixatives
Change the charges on the ionizable side chains and therefore, disrupt
electrostatic and hydrogen bonding.

 Acetic acid coagulates nucleic acids but not proteins.

 Trichloroacetic acid precipitates proteins and extracts nucleic acids.

 Picric acid forms salts with basic groups of proteins, causing them to
coagulate.
 Cross-linking Fixatives

 Aldehydes: most commonly used, such as formaldehyde

and glutaraldehyde.

 Metal salts: mercuric chloride, zinc chloride.

 Other metallic compounds: osmium tetroxide.

 Formaldehyde Fixation

 10% neutral buffered formalin (NBF): Most commonly

used fixative in diagnostic pathology.

 Pure formaldehyde is a vapor. When dissolved in water, it

forms a 37-40% solution known as “formalin”.

 “10% formalin” commonly used in routine fixation is a

10% solution of formalin or 4% formaldehyde.
 Formaldehyde Mode of Action
 Formaldehyde reacts with amino acids and proteins in the cytoplasm, nuclear
proteins and nucleic acids in the nucleus, and with unsaturated lipids.

 No interaction with carbohydrates.

 Formaldehyde reacts with different side chains to form reactive

hydroxymethyl side groups forms the initial process of covalent addition
(primary reaction in short fixation times).

 Formation of these hydroxymethyl groups denatures macromolecules and

renders them insoluble.
 Mercuric Chloride
 Used to fix bloody specimens and hematopoietic tissues (bone marrow,
lymph nodes, spleen).

 Diminishing application due to:

 Safety and health issues that arise with mercury use.

 Formation of intensely black mercury precipitates.

 Compound Fixatives
 Compound fixatives are mostly comprised of formaldehyde with other agents.

 Metallic ions have been used to aid in fixation, most prominently being
mercury, lead and zinc.

 Ethanol in combination with formaldehyde produces alcoholic formalin

Better preservation of glycogen and less shrinkage.

Good for fatty tissues (esp. breast) since it clears and extracts lipids,
thereby allowing for lymph node detection.
 Glutaraldehyde Fixation
 Glutaraldehyde has two aldehyde groups and can form
stronger crosslinks better preservation of ultrastructure.
 Less is known about mode of action.
 Not as stable as formaldehyde – requires storage at 4°C
and a pH of 5
 Disadvantages:
 Slow penetration of fixative due to strong crosslinks (tissue
must be < 0.5mm)
 Negative impact on immunohistochemistry
 Osmium Tetroxide Fixation
 Uses:
 Very important as a secondary fixative for electron microscopy
(reacts with lipids and phospholipids in cell membranes)
 Lipid stain in frozen sections.
 Disadvantages of osmium fixation:
 Toxic.
 Volatile and can readily fix nasal mucosa and the conjunctiva of the
eyes.
 Causes clumping of DNA and larges losses in proteins and
carbohydrates.
 Limited penetration into tissues.
 Factors Affecting Fixation
 Buffers and pH.
 Duration of fixation and size of specimens.
 Temperature of fixation.
 Concentration of fixative.
 Osmolality and ionic composition of fixative.
 Additives.
 Buffers and pH
 Fixation is best carried out close to neutral pH, in the
range of 6–8. Hypoxia of tissues lowers the pH, so there
must be buffering capacity in the fixative to prevent
excessive acidity.

 Common buffers include phosphate and bicarbonate.

Commercial formalin is buffered with phosphate at a pH
of 7. Unbuffered 4% formaldehyde is slightly acidic.
 Fixing in unbuffered 4% formaldehyde results in:

 Reduced formation of reactive hydroxymethyl groups and

cross-linking (can be good for immunohistochemistry).

 Artifact known as formalin pigment or hematin (brown-black

insoluble precipitate produced from hemoglobin by the action
of acid formaldehyde). This must be taken in consideration
when dealing with patients that have hemoglobin breakdown
secondary to hematopoietic diseases.
Duration of Fixation and Size of
Specimens
d = k√t
 Depth (d) reached by a fixative is directly proportional to the square
root of duration of fixation (t).

 The constant (k) is the coefficient of diffusability. It represents the

distance in millimeters that the fixative has diffused into the
tissue in one hour.
 10% formalin – 0.79
 100% ethanol – 1.00
 3% potassium dichromate – 1.33
 1 hour – 0.8 mm 2 hours – 1.2 mm 4 hours – 1.6 mm 8 hours – 2.2mm

A composite photograph showing the rate at which 10% NBF penetrates into 25 mm
cubes of liver. At the end of each time period a cube has been sliced to reveal the
advancing fixation front.

This emphasizes the importance of using small pieces of tissue

for processing. Small is good - Big is bad!
Source: http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-2-factors-influencing-chemical-fixation-formaldehyde-and-glutaraldehyde/
 During fixation with compound fixatives the most rapid
penetrator will determine the fixation image.
Example: Potassium Dichromate + 10% formalin

Potassium dichromate penetrates twice as fast as formalin

Primary fixation by potassium dichromate

Secondary fixation by formalin

 Bloody gross specimens should be washed with saline prior to
being put into fixative since some proteins in blood can
inactivate fixatives.

 The fixative volume should be at least 10 times the volume of

the tissue specimen for optimal, rapid fixation. Alternatively,
frequent changing of smaller amounts of the fixative can be
used to avoid exhaustion.

 Gross specimens should not rest on bottom of container. This

is to ensure penetration of fixative from all directions.
Agitation can also be used to enhance fixation.
 Temperature of Fixation
 Formaldehyde penetrates tissue at a faster rate at higher
temperatures.

Why?

 The diffusion of molecules increases with rising

temperature due to their more rapid movement and
vibration.
 Concentration of Fixative
 Optimum fixative concentration is determined by the solubility and
effectiveness of each fixative individually.

 Formalin: concentrations above 10% result in increased hardening,

shrinkage and the formation of a white precipitate.

 Ethanol: concentrations below 70% do not remove free water from tissues
efficiently.

 Glutaraldehyde: lower concentrations (0.25%) can penetrate tissues much

better and quicker than strong solutions (4%).
Osmolality and Ionic Composition of
Fixative
 Hypertonic solutions lead to shrinking.

 Hypotonic solutions lead to swelling.

 Some ions (Na+, K+, Ca+, Mg+) can affect cell shape and
structure. Therefore, ionic composition of fluids should be
as isotonic as possible to the tissues.
 Additives
 Certain electrolytes/non-electrolytes can improve
morphology.

 Examples of electrolytes: calcium chloride, potassium

thiocyanate, ammonium sulfate, and potassium dihydrogen
phosphate.

 Examples of non-electrolytes: sucrose, dextran, and

detergent.
 Fixation for Eye Specimens

 Entire globe is fixed in NBF for 48 hours.

 1-2 small windows can be cut into globe after 24 hours.

 After gross description, the iris and optic nerve are sliced off
(anterior and posterior)--.

 Further fixation of the remaining globe for 48 hours or more.

 Fixation of Brain Specimens

 A balance between firmness for sectioning and

responsiveness to immunohistochemistry.

 Conventional fixation of whole brain needs at least 2

weeks.

 Perfusion fixation via the middle cerebral arteries can

achieve similar results in 5-6 days.
 Fixation of Breast Specimens

 Specimens should be placed in sufficient fixative as soon

as possible to prevent loss of labile biomarkers such as
estrogen and progesterone receptors.

 10% NBF routinely used. Minimum 6-8 hours, maximum

of 72 hours.
 Fixation of Lung Specimens

 Lung biopsies: NBF

 Whole lungs from autopsies: Trachea/major bronchi used

to inflate lungs and distribute NBF. Lungs can be removed
within 2 hours and sectioned for further overnight fixation.
 Fixation of Lymphoid Tissue

 Quick transition from collection to fixation due to the

possible presence of microorganisms in the lymphoid
reticular system.