Fixation of tissues Histopathology

1. FIXATION OF TISSUES
SUNIL KUMAR.P
Haematology & Transfusion Medicine
St.John’s Medical College
Bangalore.
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2. • Definition
• Aims of Fixation
• Ideal Characteristics of fixatives
• Types of fixatives
• Mode of action
• Preparation of fixatives and indications
• Factors affecting fixation
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3. Introduction
• As soon as cells or tissues are removed from
the body they begin to die and undergo post-
mortem changes.
• These changes may be autolytic or
putrefactive.
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4. • Autolysis : Is a self destructive process due to
the release of autolytic enzymes from the
dead cells .
• Putrefaction : Occurs due to the action of
bacteria that invade the tissue.
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5. Definition of Fixative
• A fixative may be defined as a substance
which prevent post mortem changes and
preserves the morphological and chemical
characteristics of cells and tissues.
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6. Aims of Fixation
• 1.It should prevent autolysis & putrefaction of the cell.
• 2. It should penetrate evenly and rapidly.
• 3. It should harden the tissues
• 4. Increase the optical differentiation of cells & tissues
• 5. Should not cause shrinkage or swelling of the cells
• 6. Must not react with the receptor sites & thus must
notinterefere with the staining procedure.
• 7. It must be cheap and easily available.
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7. • Good fixative is most important factors in the
production of satisfactory results in
histopathology.
• Following factors are important:
• Fresh tissue
• Proper penetration of tissue by fixatives
• Correct choice of fixatives
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8. • No fixative will penetrate a piece of tissue
thicker than 1 cm.
• For dealing with specimen thicker than this,
following methods are recommended:
• 1. Solid organ:
• Cut slices as necessary as but not thicker than
5 mm.
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9. • 2.Hollow organ:
• Either open or fill with fixative or pack lightly
with wool soaked in fixative.
• 3.Large specimen:
• It requires dissection, Inject fixative along the
vessels or bronchi as in case of lung so that it
reaches all parts of the organs.
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10. Properties of an Ideal Fixative
• Prevents autolysis and bacterial
decomposition.
• Preserves tissue in their natural state and fix
all components.
• Make the cellular components insoluble to
reagent used in tissue processing.
• Preserves tissue volume.
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11. • Avoid excessive hardness of tissue.
• Allows enhanced staining of tissue.
• Should be non-toxic and non-allergic for user.
• Should not be very expensive.
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12. Mechanism / Action of Fixatives
• Most fixatives act by denaturing or
precipitating proteins which then form a
sponge or meshwork, tending to hold the
other constituents
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14. Mechanism / Action of Fixatives
• At the molecular level, fixative have the
property of coagulating proteins in the tissue,
through the formation of crosslink's between
protein molecules thereby keeping their
relation to each other.
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15. Methods of fixation
• Heat fixation
• Perfusion
• Immersion
• Vapour method
• Phase partition method
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17. Classification o f Fixatives
• 1)Physical Method of fixation.
• 2)Chemical method of fixation.
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18. Physical method of fixation
• Heat fixation :
• The simplest form of fixation is heat.
• Microwave Fixation:
• Microwave heating speeds fixation and can
reduce times for fixation of some gross
specimens and histological sections from
more than 12 hrs to less than 20 min.
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19. • Freeze-Drying and freeze substitution :
• Freeze-Drying is a useful technique for
studying soluble materials and small
molecules; tissues are cut into thin sections,
immersed in liquid nitrogen, and the water is
removed in a vaccum chamber at -40oc .
• The tissue can be post-fixed with
formaldehyde.
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21. Chemical Fixation
• Chemical fixation utilizes organic or non-
organic solutions to maintain adequate
morphological preservation.
• Chemical fixatives can be considered as
members of three major categories..
• 1.coagulant
• 2.Cross-linking
• 3.Compound fixatives
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22. Simple Fixatives
• Formalin
• The most commonly used fixative is Formalin .
• It is prepared by mixing 40 % Formaldehyde
gas in 100 w/v of distilled water.
• The resultant mixture is 100 % Formalin.
• Routinely, 10 % formalin is used which is
prepared by mixing 10 ml of 100 % formalin in
90 ml of distilled water.
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23. MECHANISM OF ACTION
• It forms cross links between amino acids of
proteins thereby making them insoluble.
• It fixes 4 mm thick tissue in 8 hours .
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24. • ADVANTAGES :
• 1.Rapid penetration
• 2. Easy availability & Relatively cheap(Low cost)
• 3. Does not over harden the tissue
• 4. Fixes lipids for frozen sections
• 5.It is relatively easy to prepare
• 6.It allows subsequent use of most staining
procedures.
• 7.frozen sections can be made with formalin fixed
tissue.
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25. DISADVANTAGES:
• 1.Irritant to the nose, the eyes and mucous
membranes
• 2. Formation of precipitate of paraformaldehyde
which can be prevented by adding 11- 16 %
methanol.
• 3. Formation of black formalin pigment , Acid
formaldehyde hematin.
• 4.It causes shrinkage of collagen.
• 5.it suspected to contain cancer producing agents
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26. Other Simple Fixatives
• Glutaraldehyde
• Osmium Tetraoxide
• Pottasium Dichromate
• Mercuric Chloride
• Picric acid
• Zenker's fluid
• Zenker’s Formal (Helly’s Fluid)
• Bouin’s Fluid
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27. Compound Fixatives
• Microanatomical fixatives:
• These are used to preserve the anatomy of
the tissue.
• Cytological fixatives:
• These are used to fix intracellular structures.
• Histochemical fixatives :
• These are used to demonstrate the chemical
constituents of the cell
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28. Microanatomical Fixatives
• 10 % Formal saline :
• It is a microanatomical fixative.
• Ideal for fixation of brain.
• Buffered formalin:
• Due to the presence of buffer, the pH of the
solution remains at neutral or near neutral.
• As a result, Formalin pigment formation
doesn’t take place
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29. Cytological Fixatives
• Nuclear fixatives :
• Carnoy’s Fluid
• Clarke’s Fluid
• Newcomer’s Fluid
• Flemming’s Fluid
• Cytoplasmic Fixatives :
• Champy’s Fluid
• Regaud’s Fluid
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30. Special Fixatives
• Dichromate and chromic acid fixatives
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31. Histochemical Fixatives:
• Formal saline
• Cold acetone
• Absolute alcohol
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32. Composition & Preparation of
Fixatives:-
• 1- 10%Formalin Solution :
• It is recommended for fixation of general surgical
biopsy specimen and tissues from CNS.
• It causes even fixation and very little shrinkage
because of its isotonicity.
• Composition & Preparation
• Formaldehyde (37-40%) - 10 ml
• Distilled water - 90 ml
• Mix well.
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33. 10% Neutral Buffered Formalin
Solution:
• It is recommended for research specimens but may also be
used for surgical and post mortem specimens.
• The special advantage of NBF is that it prevent the
formation of troublesome acid formalin pigment .
• Disadvantage : Laborious and time consuming
• Composition & Preparation
• Formaldehyde (37-40%) - 100 ml
• Distilled water - 900 ml
• NaH2PO4 - 4.0 g
• Na2HPO4 (anhydrous) - 6.5 g
• Mix to dissolve.
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34. Zenker's Solution( Mercuric Chloride
Fixative)
• M.C. Fixatives rapidly penetrates tissues and permit
excellent staining of nuclei and connective tissues.
• Disadvantage : causes hardening of tissues
• Composition & Preparation :
• fixation time 4-24 hours.
• Distilled water - 950ml
• Potassium dichromate - 25g
• Mercuric chloride - 50g
• Glacial acetic acid - 50g
• Fixed tissue should be washed overnight in running tap
water before processing.
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35. Bouin's fluid
• fixation time 6 hours.
• Composition & preparation
• Saturated aqueous solution of picric acid - 75ml
• Formalin (~ 40% aqueous solution of
formaldehyde) - 25ml
• Glacial acetic acid - 5ml
• Fixed tissue should be transferred to 70% alcohol.
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36. Carnoy's fluid
• fixation time 1-3 hours.
• Composition & Preparation:
• Ethanol - 60ml
• Chloroform - 30ml
• Glacial acetic acid - 10ml
• Fixed tissue should be processed immediately
or transferred to80% alcohol.
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37. Formal Zenker's(Helly's fluid)
• By adding formalin to Zenker’s solutions the
beneficial effects of both fixatives are
combined, minimising their disadvantages.
• Staining of nucleus, cytoplasm and connective
tissue is good with Helly’s fluid.
• fixation time 12-24 hours.
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38. Formal Zenker's(Helly's fluid)
• Composition & Preparation:
• Potassium dichromate - 25g
• Mercuric chloride - 50g
• Sodium sulphate - 10g
• Distilled water - 1000ml
• Stock solution - 100ml
• Formalin (~ 40% aqueous solution of
formaldehyde) - 5ml
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39. Factors affecting fixation:-
• 1 – Temperature
• 2 – Size of the specimen
• 3 - Volume ratio
• 4 – Duration of Fixation
• 5 - Choice of fixatives
• 6 – Penetration
• 7- Tissue Storage
• 8 –Buffer & pH
• 9 - Osmolality
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40. Fixatives used for Electron
Microscopy
• Glutaraldehyde
• Osmium tetroxide
• Formaldehyde & Glutaraldehyde mixture
• Acrolein
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41. •
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