This document discusses tissue fixation in pathology. It begins by describing the overall tissue processing steps and importance of fixation. The main types of fixatives are then outlined, including coagulant, cross-linking, and compound fixatives. Formalin and glutaraldehyde fixation are discussed in depth. Key factors that influence fixation quality like buffer pH, fixation duration, tissue size, and temperature are also summarized. The document provides a comprehensive overview of fixation in pathology.

1. Fixatives
Aseem Jain
Resident of Pathology

2. Tissue processing
• After removal from the body the tissues are exposed to a
series of reagents that fix, dehydrate, clear, and infiltrate the
tissues.
• The tissue is finally embedded in a medium that provides
support for microtomy.
• Every step of the tissue processing is important.
• Preserving cells and tissue components with minimal
distortion is the most important aim of processing tissue
processing .

3. Fixation
• This is a process by which constituents of cells and tissue are
fixed so that they can withstand subsequent treatment with
various reagents with minimum loss of architecture.
• The reagents by which this process is achieved are known as
‘Fixatives’.
• The major objective of fixation in pathology is to maintain
clear and consistent morphological features.
• Without attention to this process, the range of tests
performed in a modern histopathology laboratory will be
rendered ineffective and practically useless.

4. Why to fix.??
1. To make the cells and tissue capable to handle the
subsequent steps in tissue processing.
2. To prevent bacterial degradation of tissue.
3. To ensure good staining of the tissue.
4. To prevent further autolysis of the tissue by inactivating the
lysosomal enzymes.
5. To make the tissue useful for various special procedures like
IHC etc.

5. A Good Fixative…
1. Supports high quality and consistent staining with H&E.
2. Prevents short- and long-term destruction of the micro-
architecture of the tissue.
3. Inanimates infectious agents.
4. Is less toxic and less inflammable.
5. Permits the recovery of macromolecules (proteins,mRNA,DNA)
without extensive biochemical modifications.
6. Is useful in a wide variety of tissues.
7. Penetrates & fixes tissues rapidly .
8. Has a shelf life of at least 1 year.
9. Is compatible with modern automated tissue processors.
10. Is cost effective and easily disposable.

6. • To date, a universal or ideal fixative has not been identified.
• Fixatives are therefore selected based on their ability to
produce a final product needed to demonstrate a specific
feature of a specific tissue.
• In diagnostic pathology, the fixative of choice for most
pathologists has been 10% neutral buffered formalin.

7. Types of fixation
1. Physical fixation
• Heat fixation – Simplest form of fixation.
• Microwave fixation – Speeds fixation ; Reduces time of
fixation from >12 hours to < 20 minutes.
• Freeze drying & Freeze substitution
2. Chemical fixation
• Utilizes organic or non-organic solutions to maintain
adequate morphological preservation.
• Chemical fixatives – Coagulant, Cross linking, Compound.

8. Types of chemical fixatives
Coagulant
Fixatives
• Ethanol
• Methanol
• Acetone
• Acetic acid
• Picric acid
• Tetrachloro
acetic acid
Cross linking
fixatives
• Formaldehyde
• Glutaraldehyde
• Mercuric chloride
• Osmium tetroxide
• Chloral hydrate
Compound
fixatives
• Alcohol
formalin
• Formaldehyde -
Glutaraldehyde

9. Coagulant fixatives
• Both organic and non-organic solutions may coagulate
proteins, making them insoluble.
• Cellular architecture is maintained primarily by lipoproteins
and by fibrous proteins such as collagen; coagulating such
proteins maintains tissue histomorphology at the light
microscopic level.
• They result in cytoplasmic flocculation and poor
preservation of mitochondria so not useful for electron
microscopy.

10. Dehydrating coagulant fixatives
• Alcohols (Methanol and Ethanol) & Acetone.
• Methanol is structurally more similar to water than ethanol
; So Fixation begins at a concentration of 50-60% for
ethanol but requires a concentration of 80% or more for
methanol.
• Removal and replacement of free water from tissue by any
of these agents has several potential effects on proteins
within the tissue.

11. • Water – involved in hydrophobic as well as hydrophilic
bonding of protein.
• So if tissue is dehydrated i.e. Water is removed, the tertiary
structure of proteins is disrupted or the structure becomes
partially reversed (hydrophobic groups moving to outer
surface of proteins).
• Disruption of the tertiary structure of proteins, i.e.
denaturation, changes their physical properties, potentially
causing insolubility and loss of function.

12. • Other coagulant fixatives –
• Acidic coagulants such as picric acid and trichloroacetic acid
change the charges on the ionizable side chains of proteins
and disrupt electrostatic and hydrogen bonding.
• They also insert a lipophilic anion into a hydrophilic region
and hence disrupt the tertiary structures of proteins.
• Acetic acid coagulates nucleic acids but does not fix or
precipitate proteins; it is therefore added to other fixatives to
prevent the loss of nucleic acids.

13. Cross linking fixatives
• Formaldehyde, glutaraldehyde and other aldehydes e.g.
chloral hydrate and glyoxal, metal salts such as mercuric
and zinc chloride, and other metallic compounds such as
osmium tetroxide.
• They have actions of forming cross-links within and
between proteins and nucleic acids as well as between
nucleic acids and proteins.
• “Covalent additive fixatives”

14. Formaldehyde fixation
• Formaldehyde in its 10% neutral buffered form (NBF) is the
most common fixative used in diagnostic pathology.
• Formaldehyde is commercially supplied as a 37–40%
solution and in the following formulae is referred to as 37%
formaldehyde.
• 10% Neutral Buffered formalin -
o Tap water = 900 ml
o Formalin (37% formaldehyde solution) = 100 ml
o Sodium phosphate, monobasic, monohydrate = 4 g
o Sodium phosphate, dibasic, anhydrous = 6.5 g
• pH should be 7.2-7.4.

15. • In an aqueous solution formaldehyde forms methylene
hydrate, a methylene glycol as the first step in fixation.
• Methylene hydrate reacts with several side chains of
proteins to form reactive hydroxymethyl side groups
(–CH2–OH).
• The formation of hydroxymethyl side chains is probably the
primary and characteristic reaction.
• The formation of actual cross-links may be relatively rare at
the currently used short times of fixation.

16. • Formaldehyde also reacts with nuclear proteins and nucleic
acids.
• It penetrates between nucleic acids and proteins and
stabilizes the nucleic acid-protein shell, and it also modifies
nucleotides by reacting with free amino groups, as it does
with proteins.
• The side chains of peptides or proteins that are most
reactive with methylene hydrate, and hence have the
highest affinity for formaldehyde, include lysine, cysteine,
histidine, arginine, tyrosine, and reactive hydroxyl groups of
serine and threonine.

17. • The reactive groups may combine with hydrogen groups or
with each other, forming methylene bridges.
• If the formalin is washed away, reactive groups may rapidly
return to their original states, but any bridging that has
already occurred may remain.
• Washing for 24 hours removes about half of reactive
groups, and 4 weeks of washing removes up to 90%.
• So, in the rapid fixation used in diagnostic pathology, most
‘fixation’ with formaldehyde prior to tissue processing stops
with the formation of reactive hydroxymethyl groups.

18. • When formaldehyde dissolves in an unbuffered aqueous
solution, it forms an acid solution – Acid Formalin(pH 5-5.5)
• Acid formalin may react more slowly with proteins than NBF
because amine groups become charged.
• Acid formalin also preserves immunorecognition much better
than NBF.
• The disadvantage of using acid formalin for fixation is the
formation of a brown-black pigment with degraded
hemoglobulin.

19. Other formulae of formaldehyde –
1. Carson’s modified Millonig’s phosphate buffered formalin –
this formula is reported to be better for ultrastructural
preservation than NBF.
2. Formal (10% formalin), calcium acetate – good fixative for
preservation of lipids.
3. Formal (10% formalin), saline
4. Formal ( 10% formalin), zinc , unbuffered- is an excellent
fixative for immunohistochemistry.
5. Formalin , buffered saline
6. Formalin , buffered Zinc

20. Glutaraldehyde fixation
• Glutaraldehyde is a bifunctional aldehyde that probably
combines with the same reactive groups as does
formaldehyde.
• In aqueous solutions glutaraldehyde polymerizes, forming
cyclic and oligomeric compounds and it is also oxidized to
glutaric acid.
• To aid in stability, it requires storage at 4°C and at a pH of
around 5.

21. • Unlike formaldehyde, glutaraldehyde has an aldehyde
group on both ends of the molecule.
• With each reaction of the first group, an unreacted
aldehyde group may be introduced into the protein and
these aldehyde groups can act to further cross-link the
protein.
• Extensive cross-linking by glutaraldehyde results in better
preservation of ultra structure, but this method of fixation
negatively affects immunohistochemical methods.
• Thus, any tissue fixed in glutaraldehyde must be small (0.5
mm maximum) and, unless the aldehyde groups are
blocked, increased background staining will result if several
histochemical methods are used.

22. Osmium tetroxide fixation
• Osmium tetroxide (OsO4), a toxic solid, is soluble in water
as well as non-polar solvents and can react with hydrophilic
and hydrophobic sites including the side chains of proteins,
potentially causing cross linking.
• Osmium tetroxide is known to interact with nucleic acids,
specifically with the 2,3-glycol moiety in terminal ribose
groups and the 5,6 double bonds of thymine residues.
• Nuclei fixed in OsO4 and dehydrated with alcohol may
show prominent clumping of DNA.

23. • Large proportions of proteins and carbohydrates are lost
from tissues during osmium fixation; some of this may be
due to the superficial limited penetration of OsO4 (i.e. <1
mm) into tissues or its slow rates of reaction.
• The best characterized reaction of osmium is its reaction
with unsaturated bonds within lipids and phospholipids.
• In addition to its use as a secondary fixative for electron
microscope examinations, OsO4 can also be used to stain
lipids in frozen sections.
• Osmium tetroxide fixation causes tissue swelling which is
reversed during dehydration steps.

24. Mercuric fixatives
• Historically, mercuric chloride was greatly favored for its
qualities of enhancing the staining properties of tissues,
particularly for trichrome stains.
• Disadvantages of mercuric chloride –
1. Health and safety issues involved with the use of a mercury-
containing fixative ,
2. Reduced reliance on ‘special stains’,
3. Inevitable formation of deposits of intensely black precipitates
of mercuric pigment in the tissues. This subsequently gives
them inferior value for immunohistochemical and molecular
studies.
4. Mercury-containing chemicals are an environmental disposal
problem.

25. • Mercuric chloride reacts with ammonium salts, amines,
amides, amino acids, and sulfydryl groups, and hardens tissues.
• It is especially reactive with cysteine, forming a dimercaptide
and acidifying the solution.
• Mercury-based fixatives are toxic and should be handled with
care.
• They should not be allowed to come into contact with metal,
and should be dissolved in distilled water to prevent the
precipitation of mercury salts.
• Mercury fixatives are no longer used routinely except by some
laboratories for fixing hematopoietic tissues.

26. • Other mercuric fixatives –
1. Zenker’s solution – Just before use add 5ml of glacial acetic
acid to 95 ml of above solution. This is good fixative for
bloody(congested) specimens and trichrome stains.
2. Helly’s solution
3. Schaudinn’s solution
4. Ohlmacher’s solution
5. Carnoy- Lebrun solution- this fixative penetrates rapidly.
6. B5 fixatives- Frequently used for bone marrow , lymph nodes
,spleen , and other hematopoietic tissues.s

27. Fixatives for electron microscopy
• The preferred fixatives are a strong cross linking fixative
such as -
1. Glutaraldehyde,
2. A combination of glutaraldehyde and formaldehyde, or
3. Carson’s modified Millonig’s,
• followed by post-fixation in an agent that further stabilizes
as well as emphasizes membranes such as OsO4.

28. Fixatives for DNA, RNA & Protein analysis
1. HOPE (HEPES-glutamic acid buffer mediated Organic Solvent
Protection Effect) fixative.
2. Reversible cross-linker dithio-bis[succinimidyl propionate]
(DSP) for immunocytochemistry and expression profiling, in
addition to zinc-based fixatives.

29. Metallic ions as fixative supplement
• Several metallic ions have been used as aids in fixation,
including Hg2+, Pb2+, Co2+, Cu2+, Cd2+, [UO2]2+, [PtCl6]2+,
and Zn2+.
• Mercury, lead, and zinc are used most commonly in current
fixatives, e.g. zinc containing formaldehyde is suggested to be
a better fixative for immunohistochemistry than
formaldehyde alone.

30. Compound fixatives
• Other agents may be added to formaldehyde to produce
specific effects that are not possible with formaldehyde alone.
• Formaldehyde + Ethanol (dehydrant) = Alcoholic formalin.
• Alcoholic formalin –
1. This combination preserves molecules such as glycogen and
results in less shrinkage and hardening than pure dehydrants.
2. For fixation of some fatty tissues, such as breast, in which
preservation of the lipid is not important.
3. Fixation of gross specimens in alcoholic formalin may aid in
identifying lymph nodes embedded in fat.
4. Good at preserving antigen immunorecognition, but non-
specific staining or background staining in
immunohistochemical procedures can be increased.

31. Factors affecting quality of fixation
1. Buffer and pH.
2. Duration of fixation & Size of tissue.
3. Temperature of fixative.
4. Concentration of fixative.
5. Osmolality of fixatives and ionic composition.

32. Buffer and pH
• In a strongly acidic environment, the primary amine target
groups (–NH2) attract hydrogen ions (–NH+3) and become
unreactive to the hydrated formaldehyde (methylene hydrate
or methylene glycol), and carboxyl groups (–COO−) lose their
charges (–COOH).
• This may affect the structure of proteins.
• The extent of formation of reactive hydroxymethyl groups and
cross-linking is reduced in unbuffered 4% formaldehyde, which
is slightly acidic.

33. • At the acidic pH of unbuffered formaldehyde, hemoglobin
metabolic products are chemically modified to form a
brown-black, insoluble, crystalline, birefringent pigment.
• To avoid the formation of formalin pigment, neutral
buffered formalin is used as the preferred formaldehyde-
based fixative.
• Acetic acids and other acids work mainly through lowering
pH and disrupting the tertiary structure of proteins.
• Buffers are used to maintain optimum pH.
• Commonly used buffers - Phosphate, tris, cacodylate,
bicarbonate & acetate

34. Duration of fixation and Size of tissue
• The depth (d) reached by a fixative is directly proportional to
the square root of duration of fixation (t) and expressed this
relation as: d = k √t (k=Constant of diffusibility).
• Thus, for most fixatives, the time of fixation is approximately
equal to the square of the distance which the fixative must
penetrate.
• Gross specimens should not rest on the bottom of a container
of fixative: they should be separated from the bottom by
wadded fixative-soaked paper or cloth, so allowing
penetration of fixative or processing fluids from all directions.
• In addition, unfixed gross specimens which are to be cut and
stored in fixative prior to processing should not be thicker
than 0.5cm.

35. • Proteins inactivate fixatives, especially those in blood or
bloody fluids.
• Bloody gross specimens should therefore be washed with
saline prior to being put into fixative.
• The fixative volume should be at least 10 times the volume
of the tissue specimen for optimal, rapid fixation.
• It has been suggested that rapid fixation is acceptable as
long as histochemical staining remains adequate; and that
immunohistochemistry and other molecular techniques are
probably enhanced by shorter times of fixation using an
aldehyde based fixation.

36. Temperature of fixative
• The diffusion of molecules increases with rising
temperature due to their more rapid movement and
vibration; i.e. the rate of penetration of a tissue by
formaldehyde is faster at higher temperatures.
• Microwaves therefore have been used to speed
formaldehyde fixation by both increasing the temperature
and molecular movements.

37. Concentration of fixative
• Effectiveness and solubility primarily determine the
appropriate concentration of fixatives.
• Concentrations of formalin above 10% tend to cause
increased hardening and shrinkage.
• Ethanol concentrations below 70% do not remove free
water from tissues efficiently.

38. Osmolality of fixatives and ionic composition
• The Osmolality of the buffer and fixative is important;
hypertonic and hypotonic solutions lead to shrinkage and
swelling, respectively.
• The best morphological results are obtained with solutions
that are slightly hypertonic (400–450 mOsm), though the
osmolality for 10% NBF is about 1500 mOsm.
• Similarly, various ions (Na+, K+, Ca2+, Mg2+) can affect cell
shape and structure regardless of the osmotic effect.

39. Fixation artefacts
• During fixation, tissues commonly change in volume.
• Some intercellular structures such as collagen swell when
fixed.
• Tissues fixed in formaldehyde and embedded in paraffin wax
shrink by 33%.
• The nuclei in frozen sections are usually bigger that those of
the same tissue which has been subjected to conventional
preparation.
• Prolonged fixation in formalin can give rise to secondary
shrinkage.
• Hypertonic solutions give rise to cell shrinkage; isotonic and
hypotonic fixatives to cellular swelling and poor fixation.

40. • Artefacts related to diffusion of unfixed material:
o Diffusion of unfixed material may produce false
localization due to movement to some place other than its
original location. For example, false localization occurring
with glycogen is known as streaming artefact
• False fixation of extraneous material to tissue:
o This may occur in autoradiography with (H3) labeled amino
acids, sugars, thymidine and uridine. Tissues may
incorporate these substances into themselves by active
metabolism resulting in too high localization of various
radioactively labeled substances.

41. • Improper Fixation:
o Delay in fixation or inadequate fixation produces changes like-
a. Altered staining quality of cells
b. Cells appear shrunken and show cytoplasmic clumping
c. Indistinct nuclear chromatin with nucleoli sometimes not
seen
d. Vascular structures, nerves and glands exhibit loss of detail
e. Impression of scar formation or loss of cellularity.

42. • Use of improper fixative:
o Fixation in alcohol results in poor staining of the epithelium
and improper fixation of the connective tissue.
o Collagen bundles have an amorphous appearance that is not a
result of scar formation but rather result of artefact.
o Alcohol fixes tissues but causes severe shrinkage.
o Therefore, alcohol is not recommended as a substitute for
formalin except in extreme emergencies.
o It also makes the tissue brittle, resulting in microtome
sectioning artefacts with chattering and a Venetian blind
appearance.
o Currently, 10% neutral buffered formalin is highly
recommended for routine fixation purposes.
o One excellent indicator of poor fixation is the loss of detail of
extravasated RBC's.

43. Microtome sectioning artefact

44. • Fixation artefact simulating acantholytic disease –
o Tissue fixed in rehydrated formalin exhibits a prominent
acantholysis of superficial epithelium with preservation and
attachment of the basal cell layer to the underlying tissue.
o This acantholytic artefact simulates Pemphigus, Hailey-
Hailey disease or Darier's disease.
o Tissues allowed to air-dry will dehydrate, particularly if
placed on an adsorbent surface such as gauze sponge.
o Such tissue cannot be reconstituted and will show
dehydration artefact.

45. Cytological Fixation & Fixatives
• Rapid fixation of smears is necessary to preserve cytologic
detail of cells spread on a glass slide that are to be stained
by the Papanicolau method.
• If smears are allowed to air-dry prior to fixation, marked
distortion of the cells occurs.
• Solution of Ether + 95% ethanol – fixative of choice in past.
• Subsequently, it has been necessary to abandon this
original and excellent fixative because ether presents a fire
hazard.
• Ninety-five percent ethyl alcohol (ethanol) is now
employed as a fixative by most laboratories, with excellent
results.

46. • Smears should remain in the 95% ethyl alcohol fixative for a
minimum of 15 minutes prior to staining.
• However, prolonged fixation of several days or even weeks will
not materially alter the appearance of the smear.
• EQUIVALENT CONCENTRATIONS OF SEVERAL ALCOHOLS FOR
PURPOSES OF CELL FIXATION
1. 100% Methanol
2. 95% Ethanol
3. 95% Denatured alcohol
4. 80% Propanol
5. 80% Isopropanol
• Wet fixation with alcohol is recommended for all
nongynecologic material to be stained by the Papanicolau
method.
• For gynecologic material, coating fixatives may be used.

47. Coating fixatives
• A number of agents on the market today can be sprayed or
applied with a dropper to freshly prepared smears, thus
eliminating the use of bottles and fixing solutions.
• Most of these agents have a dual action in that they fix the
cells and, when dry, form a thin, protective coating over the
smear.
• These fixatives are particularly helpful if the smears must
be mailed to a distant cytology laboratory for evaluation.
• The method is not recommended for smears prepared from
fluids within the laboratory.

48. • As in any good method of fixation, the coating fixative
should be applied immediately to fresh smears.
• The distance from which the slides are sprayed with an
aerosol fixative affects the quality of the cytologic detail.
• Danos-Holmquist tested several spray fixatives and found
that the distance of 10-12 inches was optimal.
• Aerosol sprays are not recommended for bloody smears
because they cause clumping of erythrocytes.

49. • Coating fixatives may also be prepared inexpensively within the
laboratory. Two such methods are –
1. Polyethylene Glycol (Carbowax) Fixative
• 95% Ethyl alcohol = 50 ml
• Ether* = 50 ml
• Polyethylene glycol = 5 g
• Freshly made smears are placed on a flat surface and the
slides are covered immediately by five or six drops of the
fixative. Allow the slide to dry for 5 to 7 minutes or until an
opaque, waxy film forms over the surface.
2. Diaphane Fixative
• 95% ethyl alcohol (3 parts) + Diaphane(2 parts)

50. • Unless removed prior to staining, all coating fixatives will
contaminate the staining solutions, particularly the
hematoxylin.
• The water-soluble coating fixatives should be removed
prior to staining by maintaining two separate dishes of 95%
ethyl alcohol and leaving the slides in each dish for 5 to 10
minutes.
• The 95% ethyl alcohol used for washing off the coating
fixative should be filtered or changed at least once each
day, the number of times depending on the number of
slides that are washed.

52. Special purpose fixatives
1. Neutral buffered formalin
2. Bouin’s solution -
• 1.2% (saturated) aqueous picric acid = 750 ml
• 37% to 40% Formaldehyde solution = 250 ml
• Glacial acetic acid = 50 ml
3. Methanol acetic acid fixative -
• Equal volume of 20:1 methanol and acetic acid.
• Used when both cytologic evaluation and flow cytometry is
desired on the same urine or bladder washing sample.

53. 4. Balanced salt solutions / Normosol -
• Normosol is an excellent, low-cost alternative for short-term
storage of FNA samples.
5. Formol alcohol
6. Saccomano’s fixative –
• 50% alcohol + 2% Carbowax 1540
• Carbowax infiltrates and occupies submicroscopic spaces,
preventing cell collapse, and thus protects the cells during air
drying
• first used by Saccomanno for prefixation of sputum but can
be used for fluid specimens from other sites.

54. 7. Carnoy’s fixative –
• 95% Ethanol = 60 ml
• Chloroform = 30 ml
• Glacial acetic acid = 10 ml
• This fixative will hemolyze red blood cells and, therefore, is
useful for bloody specimens.
• However, shrinkage of the epithelial cells is greater than that
observed in specimens fixed in 95% ethanol.
• Nuclear chromatin will be lost if the cell sample remains in
Carnoy's fixative for longer than 15 minutes.
• This fixative must be prepared fresh when needed and
discarded after each use.
• Carnoy's fixative loses its effectiveness on standing, and the
chloroform can react with acetic acid to form hydrochloric
acid.

55. References
1. Bancroft's Theory and Practice of Histological Techniques,
7th Edition.
2. Koss' Diagnostic Cytology and Its Histopathologic Bases,
5th Edition.
3. Chatterjee, Shailja. “Artefacts in Histopathology.” Journal of
Oral and Maxillofacial Pathology : JOMFP 18.Suppl 1 (2014):
S111–S116. PMC.

56. Thank you