Tissue fixation

Presented by

Dr. Shrikant Sonune

Guided by

Dr Ashok Patil,

Dr Shilpa Kandalgaonkar,

Dr Mayur Chaudhary,

Dr Suyog Tupsakhare,

Dr Mahesh Gabhane.

Content

• Introduction

• Function of fixative

• Methods of fixation

• Reaction of the cell(its component) with fixatives

• Simple aqueous fixatives or fixative ingredients

• Factors affecting fixation

• Effect of fixation

• References

Introduction

Introduction

• Fixation

• Tissue processing

• Sectioning

• Staining

• Staining

• Sectioning

• Tissue processing

• Fixation

Fixation : introduction

• Fixation is the complex series of chemical events and differs for the different groups of chemical substances found in tissues.

• It is most essential part of histology. Here where everything starts.

• Why?

Introduction

• Once the tissue is removed from the body it will go

through a process of self-destruction. This process

is known as autolysis.

• If tissue is left without any preservation, then a

bacterial attack will occur, the process is known as

putrefaction.

Definition :

• It is a process by which the constituents of the cells and therefore of the tissues are fixed in a physical and partly in a chemical state , so that they will withstand subsequent treatment with various reagents with minimum of loss, significant distortion or decomposition.

• The preservation and hardening of a tissue sample to retain as nearly as possible the same relations they had in the living body

Aims & objectives of fixation • To prevent autolysis and putrefaction.

• Rapid and even penetration.

• To preserve cells and tissues in a life like manner as possible.

• Elements that are to be demonstrated must remain in maximum concentration and precise localization.

• Stabilize labile elements.

• Must be rigid to allow sectioning.

• Must allow staining.

• Optical contrast must be induced for morphological examination.

• Allow long storage of tissues

METHODS OF FIXATION

1. HEAT

2. CHEMICALS

- ADDITIVE

- NON ADDITIVE

- COAGULANT

- NON-COAGULANT

(Baker’s classification)

- COAGULANT

• ALCOHOL

• ZINC SALTS

• MERCURIC CHLORIDE

• CHROMIUM TRIOXIDE

• PICRIC ACID

- NON-COAGULANT

• FORMALDEHYDE

• GLUTARALDEHYDE

• OSMIUM TETROXIDE

• POTASSIUM

DICHROMATE

• ACETIC ACID

Classification of chemical fixatives1. Aldehydes

Formaldehyde, glutaraldehyde

2. Oxidizing agents

Osmium tetroxide, potassium permanganate,

3. Proteins denaturing agents or coagulant

Acetic acid, methyl alcohol, ethyl alcohol.

4. Other cross linking agents

Carbodiimides

5. Miscellaneous

Mercuric sodium, picric acid , dye stuff.

(by bancroff)

Acc. to no. of fixatives used:

- Simple fixatives

- Compound fixatives

i) Micro anatomical fixatives

ii) Cytological

iii)Histochemical

Reaction of fixatives with Protein

Most important reactions which stabilizes proteins

by forming cross links between soluble protein &

structural protein. Ultimately providing some

mechanical strength.

Aldehydes

• Cross links are formed between protein molecules and Aldehyde group of fixative.

• Aldehydes react with the basic amino acid residues of proteins & there is an accompanying change in isoeletric point of proteins.

• This may form the basis for the of the different staining of tissues after different fixations.

• Process takes places in 2 step

1st step-small polymers are formed

2nd step small polymers cross-link

Formations of cross linkages between Aldehyde and protein is measured by changes in viscosity, mechanical strength and molecular size of protein.

Formaldehyde

• Slow reaction

• Reversible*(in first 24 hr with

excess of water)

• Not good morphological

picture

• Less effective at cross

linking

• Loss of enzyme and

immunological activity is less

Glutaraldehyde

• Rapid

• Irreversible

• Good morphological

picture

• More effective at cross

linking

• Loss of enzyme and

immunological activity

more

Oxidizing agents

• React with protein

• Forms cross-links with proteins

• Reflected by rapid increase in viscosity

• After that decrease in viscosity , that phenomenon

is known as secondary liquefaction.

• Osmium tetroxide is more reactive towards protein.

Mercuric chloride

• It reacts with histidine residues in proteins.

• Also there is production of H+ ions making solution

more acidic more efficient.

• But after fixation ultra structural preservation is

poor.

Other fixatives

• Heat fixation /microwave fixation ------ reacts with

polar side chains of proteins. This increases their

thermal energy which cause denaturation of

proteins. This brings about tissue stabilization.

Reaction of fixative with nucleic acid

• Fixation brings about change in physical or chemical state of DNA or RNA at room temperature.

• Few fixative react with nucleic acid chemically-including mercury and chromium salts.

• Heating at 45 and 65 degrees with Aldehyde fixatives, there is uncoiling of RNA and DNA respectively.

• Ethanol, methanol and Carnoy’s fixative are

commonly used. DNA is largely collapsed in

methanol and ethanol.

• Presence of salts is known to be essential for the

maximum precipitation of nucleic acid from alcohol.

Reaction of fixative with lipidsMost of lipids are labile. So lost during routine

processing. To demonstrate them frozen section or

cryostat is used.

Aldehyde fixation:

Preservation of lipoproteins (fixation of protein

counterpart. )

Eg: phospholipids which contain amino group such as

phosphotidyl ethanolamine are fixed by aldehyde.

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HgCl₂ react with highly unsaturated compound

which form complex. It also reacts with lipids known

as plasmalogen acetal phosphatides.

Additives such as tannic acid may be used for

demonstration of lipid with light microscopy.

Ultrastructural demonstration---post fixation with

osmium tetroxide.

Cholesterol may be fixed with Digitonin for

Ultrastructural demonstration.

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Reaction with Carbohydrates

Single fixative not satisfactory.

Alcoholic or picric acid fixatives preservation of glycogen which appear coarse eg: Alcoholic formaldehyde, Rossman’s solution.

Ultra structural studies gluteraldehyde is satisfactory while potassium permanganate increase image contrast.

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Tanic acid and cetyl pyrimidium have been found useful.

Additives to vehicle like Alcian blue or ruthenium red enhance glutaraldehyde fixation of glycogen and mucins.

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Reagents used in fixation

1. Aldehydes : formaldehyde, glutaraldehyde

2. Metallic: mercuric chloride, lead fixative

3. Picric acid fixative

4. Alcoholic fixative

5. Chromate fixative

6. Osmium tetra oxide fixative

7. Acetate fixative

Formaldehyde

• Powerful reducing agent.

• Most common fixative for routine fixation of biopsy specimen.

• Formalin: 40%formaldehyde gas in water.

• Forms methelene bridges between protein molecules.

• Method 4mm block - 8hrs at room temperature

4mm block - 2hrs at 45°C

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• MOST COMMONLY IS USED FIXATIVE

10% formalin consist of

• Formalin (40% formaldehyde) 10 ml

• Water 90 ml

• Neutralization is necessary due to formation of formic acid by addition of buffer to maintain pH of 7.

Buffer added: phosphate buffer

magnesium carbonate

• Protein groups involved in formation of cross links amino, imino, peptide, hydroxyl, carboxyl and sulphahydryl.

• Formaldehyde is also obtainable in a stable solid form composed of high molecular weight polymers known as paraformaldehyde .

Advantages:

Cheap, easy to prepare, relatively stable, staining without

preliminary procedures.

Good preservation of cell morphology

Good penetration properties.

Do not cause excessive hardening.

Best fixative for nervous system

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Disadvantages-

Slow fixation reaction.

Morphological details less accurate thanglutaraldehyde.

Dermatitis of hand.

Fumes irritating to nostrils.

In tissue containing blood , dark brown artifact pigment granules are formed.

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Fixative Formula Advantages Disadvantages Uses

10%formal saline

Water- 900mlNacl- 8.5gmFormalin-100ml

•Less shrinkage

•Even fixation

•Easy sectioning

•Good staining

•Slow fixative •Hard tissues

•Neurological tissues

•Gross specimen fixation.

10% formalin 40%formaldehyde-100mlDistilled water-900ml

•Prevents pigments

•Good fixation

•Good penetration

•Preserves Enzymes and organelles

•Longer time for fixation

•Routine specimen

•Used for IHC

10% buffered neutral formalin

Formalin -100mlWater - 900mlNaH₂Po₄-3.5gmNa₂HPo₄-6.5gm

•Most routine purpose•Stops formation of formalin pigment•Fixes tissue rapidly

•Loss of basophillicstaining of the cytoplasm and nucleus•Loss of reactivity of myelin to weigert iron haematoxylinmethod

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Fixative Formula Advantage Disadvantage

Calcium acetate formalin (formal calcium)

Distilled water- 90mlCalcium acetate monohydrate- 2gmFormalin - 10ml

•Buffered at pH7 by acetate•Preserves phospholipids•Less hardening or damage•Sectioned easily

•Artifacts due to calcium

Alcoholic formaldehyde

Formalin- 100ml95%alcohol- 900mlCalcium acetate-0.5gm

•Rapid Fixation •Glycogen is better preserved

•RBC are lysed

Formol calcium Formalin- 100mlDistilled water- 900ml10%calcium chloride-100ml

•Preservation of lipids •Artifacts due to calcium

Neutral buffered phenol formalin

Neutral buffered formaldehyde-100mlPhenol- 20gm

•Stops formation of formalin pigment•Fixes tissue rapidly

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Glutaraldehyde

• Introduced by Sabatini, Bensch and Barrett

• It is a dialdehyde.

• Stable in acid solution: in pH 3 to 5

at 0 ° to 4° C

• Used in electron microscopy with osmium

tetraoxide.

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• Fixation of small tissue: 2.5% solution for 2-4 hrs at

room temperature

• Fixation of large tissue: 4% solution

for minimum 6-8hrs

fully fixed for 24hrs

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Advantages:

Better preservation of cellular and fluid proteins than

formaldehyde

More stable cross linkages

More rapid fixing action than formalin.

Less shrinkages than formalin

Give better section of blood clot and brain

Does not corrode metal

More pleasant and less irritating

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Disadvantages

More expensive

Less stable

Penetrates tissue more slowly than formalin

Inferior to formalin for PAS technique.

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Metallic fixative

mercury

• Mercuric ions act chiefly by combining with the

acidic group of proteins and strong combination with

sulfur thiol radicles.

Advantages:

Better staining of nuclei and connective tissue.

Give best results with metachromatic staining

Routine fixative of choice for preservation of detail

of photography.

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Disadvantages

Corrode all metal except nickel alloy.

Solution deteriorates rapidly.

Reduce amount of demonstrable glycogen.

Penetration is slow.

Long time fixation results in unduly hard and brittle tissue.

Diffuse black granules are seen in tissue fixed with HgCl₂.

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Picric acid fixative

• It reacts with histone and basic proteins and forms

crystalline picrates with amino acid.

• It preserves glycogen well.

• Disadvantage:

Considerable shrinkage of tissue.

It dyes the tissue - yellow colour.

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Alcoholic fixative

• Mechanism of action: alcohol denatures and

precipitate protein, possibly by disrupting hydrogen

and other bonds.

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Fixative Type Formula Advantage Disvantage Use

Ethanol and methanol

Cytological

Cytoplasmic

fixative

Ethyl alcohol and

Methyl alcohol

Rapid penetration

•Inflammable

•Causes

Shrinkage and hardens

•Smears

Glycogen

Carnoy’s

fixative

Cytological

Nuclear

fixative

Abs.Alcohol-60ml

Chloroform-30ml

Glacial acetic acid-10ml

•Excellent

Nuclear fixation and

•Rapid penetration

•Destroys

Cytoplasmic

Elements &

lipids

• Glycogen

Preservation

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Fixative Formula Type Advantages Use

Clarke’s

Fixative

•Abs.alcohol-75ml

•Glacial Acetic acid-25ml

Cytological

Nuclear

fixative

•Good penetration and nuclear details

•preserves cytoplasmic elements

Smears and chromosome study.

Alcohol formalin

95%ethanol

Formalin-10ml

Useful fixative for sputum

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Chromate fixative

• Chromium salts in water form Cr-O-Cr complexes

which have affinity for acidic and hydroxyl group of

proteins so that complexes between adjacent

protein molecules are formed.

• This leads to disruption of internal salt linkages of

protein ,thereby increasing the reactive basic

groups and enhancing acidophilia in staining.

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Fixative Formula Type Advantage and use Disadvantage

Orth’sfluid

2.5%potassium dichromate-100mlSodium sulphate-1gmJust before using,formalin-10ml

Cytoplasmicfixative

Regaudsfluid

Potassium dichromateJust before use,formalin-20ml

Cytoplasmicfixative

Demonstration of,RBC colloid containing tissue,preserve phospholipid

•Solution darken on standing•Prolonged fixation tend to bleach all tissue pigment.•Contraindicated in carbohydrates•Decrease intensity of PAS reaction.

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Osmium Tetraoxide• It is highly reactive substance , being easily

reduced.

• It gels protein probably by a process of bridge

formation between compounds.

• With lipid it forms mono and diester linkages which

are then rendered insoluble and non extractable by

fat solvent such as alcohol and xylene.

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Osmium tetraoxide

• Rapid fixing agent

• Stains tissue structure in a additive way as a grey

black deposit.

Fixative Formula Type Advantages Disadvantage Use

Flemming’sfixative:

1%aquaous chromic acid-15ml

2%aquaous osmiumtetraoxide-4ml

Acetic acid -1ml

Nuclear fixative

•It is expensive.Penetration is slow.•Difficult to counterstain.•Cause reversal of tissue acidophilia

•In electron microscopy

Champy’sfluid

3% Potassium dichromate-7ml

1% Chromic acid -7ml

2% Osmium tetraoxide -4ml

Cytoplasmicfixative

Preserves mitochondria, fat, yolk, lipids

•Needs to be freshly prepared

•Preferred for mitochondria

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EFFECT OF FIXATION

RULE #1 IS THAT FIXATIVES DENATURE MACROMOLECULES; FIXATION CHANGES THE SHAPE OF LARGE MOLECULES. THIS RULE IS THE BASIS FOR THE VARIED FUNCTIONS OF FIXATION AND WHY FIXED SPECIMENS LOOK THE WAY THEY DO UNDER THE MICROSCOPE.

• RULE #2 IS THAT DIFFERENT FIXATIVES PRODUCE THEIR OWN MORPHOLOGICAL PATTERNS. THAT IS AN OBJECTIVE FACT THAT DOES NOT IMPLY GOOD OR BAD. WHETHER WE LIKE WHAT WE SEE IS A SUBJECTIVE MATTER PREDOMINANTLY BASED ON OUR INDIVIDUAL TRAINING. MANY CHEMICALS ACT AS FIXATIVES IN THAT THEY DENATURE MACROMOLECULES, BUT FEW PRODUCE

RULE #3 IS THAT FIXATION IS A CHEMICAL REACTION THAT IS NOT INSTANTANEOUS. ITS RATE IS DEPENDENT UPON THE CHEMICAL NATURE OF THE FIXATIVE SOLUTION AND ITS TEMPERATURE.

Freida L. Carson

Factors affecting fixation.

Hydrogen ion concentration

Temperature

Penetration

Osmolality

Concentration duration

Other factors:

Volume changes

Substances added to vehicle

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Hydrogen ion concentration

• Satisfactory fixation occurs between pH 6 to 8.

• Stabilization of tertiary and quaternary structure of proteins

• By addition of acids pH decreases destruction of proteins and cause precipitation.

• Hence, fixatives must be neutralized by adding buffer.

59

Commonly used buffer system are : Phosphate, s-collidine, veronal acetate, Tris and cacodylate.

•pH chosen must be as near the biochemical optimum as possible.

•For electron microscopy , tissue must be fixed with a gradually increasing pH

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Temperature

High temperature

Rapid fixation reactions favors fixation.

Fixation should be carried out at gradually increasing temperatures

Disadvantages : 1. Risk of tissue distortion

2. Deleterious effect on

certain antigen.

Use : 1.Rapid fixation of urgent biopsy

specimen.

2.To fix tissue with tuberculosis formaldehyde at 100°C is used.

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Low temperature

Low temp. Slows down Autolysis more accurate

details.

Ultra structure and enzyme histochemistry and electron

microscopy , temp. range of 0 – 4 degrees is required.

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Penetration

• Fixation depends on diffusion of fixative into the tissue.

• Penetration of fixatives is a slow process.

• Size of specimen is important to ensure complete penetration of fixatives.

• Small or thin slices of blocks - satisfactory fixation

• Large blocks of specimen - slow fixation 63

• Slow rate of diffusion and reaction give rise to various

zones of tissue fixed to different degrees.

• d=k √t (d-depth penetrated , t-time , k-coefficient of

infusibility.

• Fixed tissue acts as a barrier to subsequent inward

diffusion of fixatives.

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Osmolality

• Hypertonic solutions - cell shrinkage.

• Isotonic and hypotonic solutions - cell swelling

• In general fixatives that act mainly on protein

precipitants cause shrinkage irrespective of what

the osmotic pressure is and for non protein

precipitants, reverse is true.

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• By varying the Osmolality, structure of membrane system

within various cells can be altered.

Thus , additives to fixatives can alter extracellular space in

tissues.

• Sucrose is commonly added to osmium tetroxide for ultra

structural studies

• Fixative solutions must be preferably isotonic, thus

cell swelling is compensated by processing and wax

impregnation.

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Concentration

• Low concentration of fixative with neutral pH favors fixation.

• Glutaraldehyde solution is used as 3% solution but it is effective even at concentration as low as 0.05% with correct pH of fixative.

• Presence of buffer causes polymerization of Aldehyde with a consequent decrease in effective concentration.

• Staining of tissue is altered with the concentration of fixative employed.

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Duration• Long duration

- In Aldehyde :

- a) inhibit enzyme activity and immunological reactions

- b) shrinkage and tissue hardening.

• Glutaraldehyde

• longer duration of fixation

• effective polymer formation

• advantageous.

- In oxidizing fixatives : degrade the tissue by oxidative cleavage of proteins and loss of peptides.

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i) Changes in volume-

Ideally, changes in processing and fixation cancel each other giving no net change.

Formalin fixed tissues along with paraffin embedding causes

33% shrinkage .

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ii) Substances added to vehicle:

Fixative

fixative agent + Buffer + water.

Salts added have denaturing and stabilizing effect on

proteins.

Eg : NaCl2 + HgCl2 , Tannic acid, Alcian blue for

ultra structure.

Plastination

A technique or process used to preserve bodies or body parts, tissue. The water and fat are replaced by certain plastics(or resin), yielding specimens that can be touched, do not smell or decay, and even retain most properties(especially morphology) of the original sample.

References

John D. Bancroft: Theory & Practice Of Histological Techniques.

Culling’s: Histological Techniques

Fixation and Processing-Freida L. Carson