tissue fixation

1. Tissue fixation
Noor Ullah
Lecturer MLT, STMU
10/10/2018 1

2. Micro techniques
• Micro technique deals with the preparation of tissues for microscopic
examination.
• It is the study of procedures to reach the final stained slide of the
specimen.

3. Stages in Histopathology
1. Documentation
2. Fixation
3. Gross examination
4. Decalcification
5. Tissue processing
6. Embedding
7. Sectioning
8. Staining
9. Mounting

4. Type of material obtained in laboratory
• The human tissue comes from the surgery (Biopsy) and/or from the
dissection room (Autopsy).
• From surgery two types of biopsy could be obtained:
• Incisional Biopsy: A small piece of lesions or tumor is removed and
sent for diagnosis before final removal of the lesion or tumor .
• Excisional Biopsy: whole tumor or lesion is removed for examination.

5. Excision
Cell Death
Fixation
Embedding
Dehydration
Examination and
Diagnosis
Ancillary Studies
T
i
m
e
{
Nucleases
Proteases
Lipases
Saccharidases
The time between excision
and fixation can vary widely,
as can the conditions of
fixation.
Tissue Processes in Pathology

6. Proteases
• Destroy protein antigens
• Not all protein antigens are destroyed at the same rate
• Nucleases
• Destroy nucleic acids
• RNA destroyed much more quickly than DNA
• Found in different levels in different tissues.
• For example, pancreas and eosinophils have extremely high levels of
ribonucleases

7. Stability
• In general DNA>Protein>RNA
• Some proteins are essentially as stable as DNA, however.
•Fixation
• Stops degradation of cellular components by enzymes
• Preserves cellular morphology

8. Definition of fixation
• Process by which the constituents of the cells and therefore of
the tissues are fixed in a physical, partly also in a chemical
state so that they will withstand the subsequent treatment
with various reagents with minimal loss , distortion or
decomposition.
• Fixation is the foundation for the subsequent stages in the
preparation of the sections

9. Why fixation is done?
• If a fresh tissue is kept at room temperature it will become liquefied with a
foul odor mainly due to action of bacteria i.e. putrefaction and autolysis so
the first and fore most aim of fixation is
1. To preserve the tissue in as life like manner as possible.
2. To prevent postmortem changes like autolysis and putrefaction.
• Autolysis :is the lysis or dissolution of cells by its own enzyme action
probably as a result of rupture of lysosomes.
• Putrefaction: The breakdown of tissue by bacterial action often with
formation of gas.

10. Why fixation is done?
3. Preservation of chemical compounds and micro anatomic
constituents so that further histochemistry is possible.
4. Hardening : the hardening effect allows easy manipulation of soft
tissue like brain, intestines etc.
5. Solidification: Converts the normal semifluid consistency of cells
(gel)to an irreversible semisolid consistency (solid).
6. Optical differentiation: Fixatives alter to varying degrees the
refractive indices of the various components of cells and tissues and
thus increase its optical differentiation.
6. Effects of staining: Certain fixatives like formaldehyde intensifies the
staining character of tissue especially with haematoxylin.

11. Properties of an Ideal Fixative
1. Prevents autolysis and bacterial decomposition.
2. Preserves tissue in their natural state and fix all components.
3. Make the cellular components insoluble to reagent used in tissue
processing.
4. Preserves tissue volume.
5. Avoid excessive hardness of tissue.
6. Allows enhanced staining of tissue.
7. Should be non-toxic and non-allergic and non corrosive for user.
8. Should not be very expensive.
9. It must penetrate the tissue rapidly and evenly
10. It must be simple to prepare and economical to use

12. Reaction Of Fixatives
1. Proteins:
 Cross links are formed between
proteins.
 Soluble proteins are fixed to structural
proteins-insoluble-mechanical strength-
allowing subsequent manipulations on
tissues
 Formaldehyde -reversible.
 Glutaraldehyde -rapid & irreversible.
 React with basic amino-acid residues

13. Reaction Of Fixatives
2. Nucleic acid:
 Fixation brings a change in the physical & chemical state of RNA & DNA.
 Uncoiling of DNA & RNA occurs with formalin when heated to 45˚C & 65˚C
respectively.

14. 3. Lipids:
Phospholipids are fixed by aldehydes.
Formaldehyde reacts with unsaturated fatty acids
hence less lipid can be demonstrated in tissue
stored in it for a long time.
Mercuric chloride reacts with lipids to form
complexes.
Ultrastructural demonstration of lipids – post fixing
in imidazole-osmium tetroxide.

15. 4. Carbohydrates:
• Carbohydrates are more water soluble- difficulty in total preservation
• They bind with fixed protein
• So the fixatives which are used for proteins, can be used for carbohydrate
preservation.
• Fixed protein traps carbohydrates.
• Glycogen not bound to protein- fixed protein form lattice around glycogen to
preserve it
• Glycogen are more demonstrable in liver cells

16. Fixation artifacts
• Fixation is associated with some artifacts:
• Formaldehyde fixatives give brown pigmentation to tissues
• Mercuric chloride fixatives leave a black precipitate in tissues
• Some fixatives produce shrinkage in tissues while some others cause
swelling.
• Due to poor penetration of fixatives macromolecules like glycogen diffuse
from unfixed parts giving false localization or loss. This is termed streaming
artifact and mostly seen in case of glycogen.

17. Factors affecting fixation
1. Buffers & hydrogen ion concentration:
 Best fixation occurs between pH 6-8
 Buffers used – phosphate, s-collidine, bicarbonate,
2. Temperature:
 Most tissues fixed– room temp
 Electron microscopy & histochemistry: 0-4˚C
3. Penetration of tissues:
 Blocks should be small or thin to ensure adequate penetration.

18. Factors affecting fixation
4. Volume changes:
 Due to inhibition of respiration, membrane permeability changes,
changes in ion transport through membrane.
 Tissues fixed in formaldehyde & embedded in paraffin shrink by
33%
5. Osmolality of fixative:
 Hypertonic solutions – cell shrinkage
 hypotonic solutions – swelling of cells & poor fixation.
 Best – slightly hypertonic solutions.

19. 6. Substances to vehicle:
 Adding substances to fulfill certain functions.
 Denaturing effects, some stabilize proteins.
 Eg. Sodium chloride & sodium sulphate used
with mercuric chloride.
 Tannic acid enhances fixation of lipids &
proteins in EM
7. Concentration of fixatives:
 Different concentrations have different
effects on morphology.
 Effects subsequent staining

20. 8. Duration of fixation:
 Formalin – 2-6hours
 Electron microscopy – 3 hours
 Formaldehyde – prolonged fixation – shrinkage &
hardening of tissue.
 Gluteraldehyde – prolonged fixation – advantageous.
 Long fixation in aldehydes - inhibits enzyme activity.
 Long fixation in oxidizing fixatives – degrade the tissue.

21. General classification of fixatives
1. Aldehydes: formaldehyde, glutaraldehyde, acrolein
2. Oxidizing agents: osmium tetroxide, potassium permanganate, potassium
dichromate
3. Protein denaturing agents: acetic acid, methyl alcohol, ethyl alcohol
4. Other cross linking agents: carbodiimides
5. Physical agents: heat, microwave, Freeze drying
6. Unknown mechanism: mercuric chloride, picric acid
7. HOPE fixatives: formalin like morphology, good protein antigenicity for enzyme
histochemistry, good for RNA & DNA yeilds

22. Baker classification
 Coagulant fixatives includes:
1. Formaldehyde
2. Gluteraldehyde
3. Osmium Tetroxide
4. Potassium Dichromate
5. Acetic Acid
 Non-Coagulant fixatives includes:
1. Alcohol
2. Zinc salts
3. Mercuric chloride
4. Chromium trioxide
5. Picric Acid

23. Classification-based on structure
1. Micro-anatomical fixatives:
When anatomy of tissues with correct relationship of tissue layers &
large aggregate of cells is to be preserved.
2. Cytological fixatives:
To preserve constituent elements of cells.
Elements being preserved at the expense of penetration, ease of
cutting & loss of other cell structures.
3. Histochemical fixatives:
Used when histochemical tests are to be applied ( IHC)

24. Micro anatomical fixatives
I. Routine formalin fixatives:
1. 10% Formol-Saline:
 10% formalin in 0.9% sodium chloride.
 Layer of marble chips/calcium carbonate added to neutralize formic
acid production.
2. Buffered Formalin:
 Formalin – 10ml
 Acid sodium phosphate monohydrate 0.4g
 Anhydrous disodium phosphate 0.65g
 Water to 100ml

25. Micro anatomical fixatives
3. Formol Calcium(Baker)
 Formalin – 10ml
 Calcium chloride – 2g
 Water – 100ml
 Chloride preserves phospholipids
4. Formol Calcium(Lillie)
 Acetate instead of chloride
 Easily prepared
 Widely used for routine fixation.

26. Mechanism of action:
• Aldehydes (cross linking agents)- act by creating covalent
chemical bonds between proteins of tissues- anchor the
insoluble compound to cytoskeleton-protect secondary as
well as tertiary structure of protein-provide mechanical
strength/ additional rigidity to tissue structure.
• Oxidizing agents- joins with various side chains of protein
molecules & other biomolecules- allow formation of cross
link- stabilizes tissue structure

27. • Protein denaturing agents: reduce the solubility of
protein without combining with it & disrupts the
hydrophobic bonds which is needed for its tertiary
structure to form.
• Mercurials (B5 fixatives): it increases the staining
brightness & give good nuclear detail. Good for reticulo-
endothelial tissue & hematopoietic tissue.
• Picrates: binds with histone & basic proteins to form
crystalline picrates with amino acid & precipitates protein.

28. FORMALDEHYDE
• Gas soluble in water up to 40% by wt.
• Available as 40% formaldehyde or formalin.
• Stabilizer – 10-14% methanol
• 10% formalin
• Acidic solution.
• On storage becomes acidic by formation of formic acid.
• Colourless.
• Turbid on keeping - paraformaldehyde.
• fixes protein, lipids well preserved.
• Favors staining of acidic structures like nuclei with basic dyes
• Diminishes effect of acid dyes on basic structures like cytoplasm.

29. Advantages:
• Formalin is cheap
• Easy to prepare
• Relatively stable
• Frozen sections can be prepared with ease.
• Staining of fat and tissue enzymes.
• Penetrates tissues well.
• Beneficial hardening with little shrinkage

30. Formaldehyde- Advantages
• Formalin is cheap
• Easy to prepare
• Relatively stable
• Frozen sections can be prepared with ease.
• Staining of fat and tissue enzymes.
• Penetrates tissues well.
• Beneficial hardening with little shrinkage
• Natural tissue colors are retained.
• Does not require washing before processing.
• Best fixative for nervous system

31. Formaldehyde- disadvantages
• unpleasant vapor irritation to eyes & respiratory epithelium
• Formalin dermatitis
• Brown, granular material, extracellular, birefringent
• Progressive in deposition
• Often seen after several days
• Action of acid formalin on blood
• Avoided by using buffered formalin.
• Removed – treatment with saturated alcoholic solution of picric acid
for 20mins

32. Formaldehyde- disadvantages
• Pink disease
• Peculiar artifact seen in sections fixed in formal saline & stained with
H&E.
• Complete or partial failure of nuclei to stain with haematoxylin – take
up eosin – loss of nuclear margin distinction.
• Lymphoid & epithelial tissue – most distinct
• Patchy distribution
• Avoided by using - 2% acetic acid in 10% formalin
• When present – treat with 1% hydrochloric acid in absolute alcohol
for 1 hour before staining with H&E.

33. GLUTERALDEHYDE
• Used for Electron Microscopy with osmium tetroxide
• Advantage:
• Most efficient cross linking agent for collagen
• More rapid fixation than formalin.
• Disadvantage:
• Poorer penetration
• False positivity with PAS
• More costly

34. MERCURIC CHLORIDE
• White crystalline substance.
• Powerful protein precipitant, fixes both nucleus & cytoplasm well
favoring its staining.
• Conjunction with other fixatives.
• Advantages: Rapidly penetrates & hardens tissue, radio-opaque
• Disadvantages: Extremely poisonous & corrosive to metals.
• Intolerant fixative.
• Pollution to environment.

35. MERCURIC CHLORIDE
• Mercury pigment – brown to black granular deposit.
• Treatment-
• Place section in 0.5% iodine in 80% alcohol for 3mins
• Rinse in water
• Place in 3% aqueous sodium thiosulphate for 3mins
• Wash in running water for 1-2mins

36. PICRIC ACID
• Bright yellow crystalline substance.
• Damped with water because of
explosive properties.
• Enhances cytoplasmic staining.
• Acts as mordant
• Much shrinkage but little hardening.

37. POTASSIUM DICHROMATE
• Orange crystalline substance
• Less acidic pH – fixes cytoplasm &
mitochondria
• More acidic pH – fixes nucleus &
cytoplasm
• Mordant
• Wash in running water after to
prevent formation of insoluble
precipitate.
• Prolonged exposure causes tissue to
become brittle.

38. OSMIUM TETROXIDE
• Pale yellow.
• Demonstrates lipid like myelin.
• Excellent preservation of detail of
single cells hence used for EM.
• Uneven penetration for pieces more
than 2-3mm
• Storage in dark, cool place
• Vapour is irritating, causes
conjunctivitis
• Uneven fixation

39. ACETIC ACID
• Colorless liquid with pungent odor.
• Used in compound fixatives
• Swells collagen fibres
• Precipitates nucleoproteins
• Solvent action on cytoplasmic granules.

40. ETHYL ALCOHOL
• Colorless.
• Powerful dehydrating agent.
• Causes shrinkage & hardening
• Coagulates protein but not nucleoprotein.
• Precipitates glycogen.
• Used in histochemical method for enzymes.

41. Fixatives for DNA, RNA, and protein analysis
• The compounds included the commercially available HOPE (Hepes-
glutamic acid buffer mediated Organic solvent Protection Effect) for
the detection of nucleic acids, in addition to zinc-based fixatives.
• In contrast to other fixation methods HOPE does not completely
denature or cross-link structural proteins, enzymes, and nucleic
acids.
• They remain in an almost native state. This means that HOPE- fixed
tissue can also include active viruses, prions, microorganisms etc.

42. Fixation for selected individual tissues
• Eyes:
• Eyes may be fixed in NBF, usually for 48 hours.
• After gross description, the anterior (iris) and posterior (e.g. optic
nerve) are removed with a new, sharp razor blade and the
components of the globe are fixed for an additional 48 hours, or
more, in buffered formaldehyde, before being processed.
• Embedding may be in celloidin or paraffin.

43. Fixation for selected individual tissues
• Brain:
• The problem of fixing a whole brain is to render it firm enough to
investigate the neuroanatomy and to produce sections to show
histopathology and to respond to immunochemistry if required.
• Conventionally this fixation takes at least 2 weeks.
• Perfusion technique is used which allows to fix brain tissue and the report
issued in 5–6 days.
• This method depends on the perfusion of the brain via the middle
cerebral arteries.
• Fixatives may also be enhanced by the use of microwave technology.
• Alcoholic formalin should not be used for fixation if immunohistochemistry
is to be performed using biotin-avidin (streptavidin) methods

44. Fixation for selected individual tissues
• Breast:
• Clinical samples should be fixed in 10% NBF for between a minimum of 6–
8 hours and a maximum of 72 hours, and should be sliced at 5mm
intervals after appropriate gross inspection and margins designation.
• Time from tissue acquisition to fixation should be as short as possible in
order to prevent lysis of clinically important biomarkers, such as estrogen
receptors, progesterone receptors and the human epidermal growth factor
receptor-2 (HER2).
• They should be placed in a sufficient volume of NBF to allow adequate
tissue penetration.
• If the tumor specimen has come from a remote geographical location, it
should be bisected through the tumor on removal and sent to the
laboratory immersed in a sufficient volume of NBF (Hammond et al., 2010).

45. Fixation for selected individual tissues
• Lungs:
• Lung biopsies are typically fixed in NBF.
• The lungs from autopsies may be inflated by and fixed in NBF via the
trachea or major bronchi.
• Gross sections are fixed overnight and sections to be processed and
cut the next day.

46. Fixation for selected individual tissues
• Lymphoid tissue:
• Special care should be taken with all lymphoid tissue, as many organisms (e.g.
Mycobacterium tuberculosis and viruses) may sequester themselves in the
lymphoid reticular system.
• The lymphoid tissue is usually sliced and a representative sample of fresh tissue
taken for special studies (e.g. flow cytometry or molecular analysis).
• The rest of the lymph node is fixed in NBF, though some laboratories fix part of
the tissue in B5 or zinc.

47. Fixation for selected individual tissues
• Testis:
• Biopsies of the testes are fixed routinely in NBF.
• Muscle biopsies:
• Biopsies of muscle are received fresh. A portion is separated for enzyme
histochemistry.
• The tissue for routine histological assessment is fixed in NBF and
embedded so the fibers of the specimens are viewed in cross-section and
longitudinally.

48. Fixation for selected individual tissues
• Renal biopsies:
• Renal core biopsies should be subdivided into three and each piece should
contain adequate numbers of glomeruli. Each portion is then preserved,
depending upon the method to be used for analysis:
• NBF for routine histology
• Buffered glutaraldehyde (pH 7.3) for ultrastructural analysis
• Sudden frozen in isopentane and liquid nitrogen for immunofluorescence
examination.