FIXATION OF BIOPSY

Awal Mir Khattak

Demonstrator MLT

B.Sc. MLT Baqai Medical University Karachi

M.Sc. Hematology Baqai Medical University Karachi
M.Phil. Medical Lab Sciences, The University of Haripur
 BIOPSY FIXATION
• Biopsy fixation is the process of tissue preservation in
which tissue survived the subsequent treatment with
various reagents with minimal loss, distortion or
decomposition.
• In this process constituents of tissue cells are fixed in a
physical and partly also in a chemical state.
• Fixation minimizing the loss of cellular components such
as proteins, small peptides, mRNA, DNA and lipids.
• It is also prevents the destruction of macromolecular
structures such as cytoplasmic membranes, smooth
endoplasmic reticulum, rough endoplasmic reticulum,
nuclear membranes, lysosomes and mitochondria.
 BIOPSY FIXATION
• If soluble components are lost from the cytoplasm of
cells, the color of the cytoplasm on hematoxylin and
eosin (H&E) staining will be modified and appearance
of the microanatomy of the tissue will be altered.

• Similarly, immunohistochemical evaluations of

structure and function may be reduced or lost.
 WHY FIXATION IS DONE
1. To prevent autolysis or Putrefaction.
2. Preservation of chemical compounds and micro
anatomic constituents.
3. Hardening effect: This allows easy manipulation of soft
tissue like brain, intestines etc.
4. Solidification effect: Normal semifluid consistency of cells
(gel)to an irreversible semisolid consistency (solid).
5. Optical differentiation: Fixatives alter refractive indices of
the various components of cells, tissues to differentiate
easily.
6. Effects of staining: Certain fixatives like formaldehyde
intensifies the staining character of tissue.
 TYPES OF FIXATION
• There is two type of biopsy fixation.
1. Physical method: Heating, microwaving and freeze-
drying
2. Chemical method:
• Coagulant fixation such as
Formaldehyde, Gluteraldehyde, Acetic Acid etc.
• Non-Coagulant fixation such as:
Alcohol, Zinc salts, Chromium trioxide, Picric Acid etc.
• Cross-linking
 PHYSICAL FIXATION
1. Heat Fixation:
• This is the simplest form of fixation. Boiling of egg
precipitates the proteins.
• On cutting yolk and egg white can be identified
separately.
• Heat fixation attaches the section to the slide and
partially fixes it by heat and dehydration.
• Even on boiling (tissue in normal saline) adequate
morphology can be obtained
 PHYSICAL FIXATION
2. Microwave Fixation:
• Microwave heating can reduce times for fixation more
than 12 hours to less than 20 minutes.
• Microwaving tissue in formalin results in the production
of dangerous, potentially explosive vapors.
• Such case must processed in biosafety cabinet.
• Commercial glyoxal-based fixatives which do not form
vapors when heated at 55°C have been introduced as an
efficient method of microwave fixation
 PHYSICAL FIXATION
3. Freeze drying Fixation:
• Freeze-drying is a useful technique for studying soluble
materials, small molecules and mostly used in research
environment and rarely in clinical set up.
• Tissues are cut into thin blocks, immersed in liquid nitrogen
and the water removed in a vacuum chamber at −40°C.
• Or Specimens are immersed in fixatives (acetone or alcohol) at
−40°C, this slowly removes water through dissolution of ice
crystals and proteins are not denatured.
• Bringing the temperature gradually up to 4°C will complete
the fixation process.
 CHEMICAL FIXATION
• This utilizes organic or non-organic solutions to maintain
adequate morphological preservation.
• Chemical fixation can be divided into three major
categories.
1. Coagulant fixation.
2. Cross-linking
3. compound
 CHEMICAL FIXATION
1. Coagulant fixation.
• Both organic and non-organic solutions may coagulate
proteins making them insoluble.
• Coagulating proteins maintains tissue histomorphology
at the light microscope level.
• Coagulant fixation result in cytoplasmic flocculation and
poor preservation of mitochondria and secretory
granules.
• These fixatives are not useful in ultrastructural analysis.
 CHEMICAL FIXATION
• Dehydrate coagulant fixation
• The most commonly used in this group are alcohols
and acetone.
• The removal and replacement of free water from tissue
this agent has several potential effects on proteins within
the tissue.
• By removing water, weakens hydrophobic bonding and
water molecules participate in hydrogen bonding in
hydrophilic areas of proteins.
• Together, these changes act to disrupt the tertiary
structure of proteins lead to tissue preserved.
 CHEMICAL FIXATION
2. Non-coagulant cross-linking fixation
• Several fixatives secondary forming cross-links
both within and between proteins and nucleic acids.
• Cross-linking may not be a major mechanism with
short times of fixation and therefore ‘covalent additive
fixatives’ may be a better name for this group.
• Examples include formaldehyde, glutaraldehyde,
aldehydes, metal salts (mercuric and zinc chloride),
• Metallic compounds (osmium tetroxide)
 CHEMICAL FIXATION
2. Compound fixatives tissue fixation
• Pathologists use formaldehyde-based fixatives to ensure
reproducible histomorphometric patterns.
• Other agents may be added to formaldehyde to produce
specific effects which are not possible with formaldehyde
alone.
• Dehydrant ethanol, for example, can be added to
formaldehyde to produce alcoholic formalin.
• This combination preserves molecules such as glycogen
and results in less shrinkage and hardening than pure
dehydrants.
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