Histopathology of fixative

1. FIXATION AND FIXATIVES
E. Adankwah, Ph.D
1

2. Background
• Specimen accession
– Tissue specimens received has request
form that lists patients information,
history along with a description of the
site of origin
– Specimens are accessioned by giving
them a unique number by which they
will be identified .
2

3. Background
3
• Gross examination
– Tissue are examined by a pathologist, pathologist assistant or
resident.
– Involves describing the specimen and placing all or parts of it
into a small plastic cassette which holds the tissue for
subsequent processes.

4. Background
4
Grossing tools
Cassettes

5. Background
5

6. Background
• When a tumour is suspected, then the specimen is often marked
with ink in order to mark the margins of the specimen.
• Different colour inks can be use to identify different areas.
• The macroscopic description is usually dictated for subsequent
secretarial transcription, or occasionally can be simply written
down for typing later.
6

7. 7

8. 8

9. Stages of tissue processing
Grossing
Surgical
9

10. Fixation
• Tissue block is taken by biopsy,
surgical excision or postmortem
• Fixation is a chemical process by
which biological tissues are preserved
from decay, either through autolysis
or putrefaction.
• Fixation terminates any ongoing
biochemical reactions, and may also
increases the mechanical strength or
stability of the treated tissues
10

11. Purpose of Fixation
• To preserve a sample of biological material (tissue or cells) as
close to its natural state as possible in the process of preparing
tissue for examination.
• To prevent postmortem changes like autolysis and putrefaction
• Preservation of chemical compounds and microanatomic
constituents so that further histochemistry is possible
• Harden the tissues, as fixation causes coagulation of proteins.
• Fixation has a mordanting effect, facilitating subsequent
staining of tissues
11

12. Classification of Fixatives:-
• Physical fixatives
– Heat
– Microwaving
– Freeze-drying and substitutes
• Chemical fixatives
** General classification; Heat, Perfusion and immersion
12

13. 13
Heat
Immersion
Perfusion

14. Formal Calcium
Alcoholic formalin
Heidenhain Susa
14

15. Mechanism of action of fixatives
• Based on their mode of action fixatives can be classified
as;
– Cross-linking fixatives: Aldehydes
– Oxidising agents
– Coagulant/Precipitating fixatives (denaturing)
– Other fixatives
15

16. Coagulant/Precipitating fixatives (1)
• Coagulating of macromolecules such as lipoproteins,
fibrous proteins (collagen) to maintain tissue
morphology.
• It act by reducing the solubility of protein molecules and
by disrupting the hydrophobic interactions which give
many proteins their tertiary structure
16

17. • Protein primary structure intact. Alters secondary and tertiary
structures
• Hydrophilic/phobic inversion often irretrievably, with loss of up
to 40% of protein.
• The most commonly used precipitating fixatives are ethanol,
methanol and acetone
• Other types of coagulant fixatives include picric acid and
trichloroacetic acid
• Acetic acid coagulates nucleic acids but not proteins.
– Used as additive to prevent nucleic acid loss
17
Coagulant/Precipitating fixatives (2)

18. • Picric acid dissolves in water to form weak acid
solution. It forms salt with basic groups of protein,
causing the proteins to coagulate
• Picric acid give brighter staining but may cause
hydrolysis and loss of nucleic acids in low pH
solutions
18
Coagulant/Precipitating fixatives (3)

19. Formulations of Picric acid fixatives
• Bouin’s solution: excellent general fixative for connective
tissue stains and fatty tissues
• Hollande’s solution: Useful of GIT biopsies and
endocrine tissue
• Rossman’s and Gendre’s fluid
19

20. Formulations of dehydrant coagulant fixatives
• Absolute Ethanol and 70–95% Ethanol
• 100% Methanol
• 100% Acetone
• Clarke's solution
– It give good nuclear detail but loss of lipids
• Carnoy's fixative: Useful for RNA stains
– It shrinks and hardens tissue, useful in cytology to lyse red blood
cells
20

21. Pitfalls of Coagulant fixatives
• Not useful for ultrastructural analysis as it causes
cytoplasmic clumping and poor preservation of
mitochondria and secretory granules.
• The alcohols are known to cause shrinkage of tissue
during fixation.
• Acetic acid alone is associated with tissue swelling;
combining the two may result in better preservation of
tissue morphology.
21

22. Cross-linking fixatives
• Mode of action: they act by creating covalent chemical
bonds between proteins in tissue
• This anchors soluble proteins to the cytoskeleton, and
lends additional rigidity to the tissue
• Examples: formaldehyde, glutaraldehyde,
• Others; metal salts such as mercuric and zinc chloride,
osmium tetroxide
22

23. Formaldehyde fixation (1)
• Pure formaldehyde is a gas, dissolve in water to form a solution
containing 37-40% formaldehyde (formalin)
– Commonly used : 10% neutral buffered formalin
• Protein secondary structure intact. Only modifies tertiary and
quaternary structures.
• Methylene bridge cross-links and mostly (90%) retrievable, with
little loss (<1%) of protein.
• Cannizzaro reaction??
23

24. 24

25. 25

26. Formaldehyde fixation (2)
• Paraformaldehyde is a polymerized form of formaldehyde,
usually obtained as a fine white powder, which depolymerizes
back to formalin when heated
• Formalin penetrate tissue well but relatively slow.
• Oxidation of formaldehyde results in an acid solution (formic
acid)
• Acid formalin leads to the formation of brown-black pigment
with degraded haemoglobin.
26

27. Formalin artefacts
27
Formalin pigments
Tissue separation by
prolonged fixation

28. Formulations of formaldehyde
• Neutral buffered 10% formalin
• Formal (10% formalin) saline
• Formal (10% formalin) zinc unbuffered
• Formal (10% formalin), calcium acetate
– Good fixative for preservation of lipids
28

29. Glutaraldehyde fixation (1)
• It does not penetrate thicker tissue specimens as
effectively as formaldehyde. Thus small blocks of tissue
is required.
• It may offer a more rigid or tightly linked fixed.
• It results in better preservation of ultrastructure but the
rate of penetration is slow.
• Suitable for electron microscopy
29

30. • It causes rapid and irreversible changes, fixes
quickly gives best overall cytoplasmic and nuclear
detail.
• Some fixation protocols call for a combination of
formaldehyde and glutaraldehyde, so that their
respective strengths complement one another.
30
Glutaraldehyde fixation (2)

31. Oxidising fixatives (1)
• Oxidizing agents include permanganate fixatives
(potassium permanganate), dichromate fixatives
(potassium dichromate), and osmium tetroxide.
• They cause extensive denaturation despite preserving
fine cell structure and are used mainly as secondary
fixatives.
• Osmium tetroxide is often used as a secondary fixative
when samples are prepared for electron microscopy.
31

32. Oxidising fixatives (2)
• Large proportions of proteins and carbohydrates are lost
from tissues during osmium fixation due slow
penetration and reaction rates.
• Can be used to stain lipids in frozen sections
• Osmium tetroxide fixation however causes tissue
swelling which is reversed during dehydration steps.
32

33. Mercuric fixatives
• Mercurials fix tissue by an unknown mechanism.
• These fixatives penetrate relatively poorly and cause
some tissue hardness, but are fast and give excellent
nuclear detail
• Mercuric pigments removed by iodine solution followed
by sodium thiosulphate
33

34. Formulations of Mercuric fixatives
• They contain mercuric chloride and include such well-
known fixatives as B-5, Zenker's and Helly solution
• Their best application is for fixation of bone marrow,
lymph nodes , spleen and other hematopoietic tissues
34

35. Characteristics of an ideal fixatives (1)
• Avoid excessive hardness of tissue
• Allows enhanced staining of tissue
• Should have good toxicological and flammability profiles
to permit safe use
• It should penetrate and fix tissues rapidly
35

36. Characteristics of an ideal fixatives (2)
• Should be cost-effective
• It must also permit the recovery of macromolecules
including proteins, mRNA and DNA without any
extensive modifications
• It should allow tissue to be stored for long time
• It must kill the cell quickly without shrinkage or
swelling
36

37. Factors affecting the quality of fixation (1)
• Buffers and pH
• Duration of fixation and size of specimens
ü Medawar, (1941) established that fixatives obey the
diffusion laws. That is, the depth penetrated is
proportional to the square root of time. Each fixative
has a unique coefficient of diffusibility, designated K.
Penetration rate may be determined from the
formula;
d = K x √t
Where d = depth in mm, K = the Medawar coefficient
√t = the square root of fixation time in hours.
37

38. ü constituents of compound fixatives will penetrate tissue
at different rates
ü The fixative volume should be at least 10-20 times the
volume of tissue specimen
ü Gross specimens, bloody/body fluid and unfixed gross
specimens??
38
Factors affecting the quality of fixation (2)

39. • Temperature
• Concentration
• Osmolality of fixatives and ionic composition
• Additives
39
Factors affecting the quality of fixation (2)

40. THANK YOU FOR LISTENING
40