Tissue Fixation and Processing

BASIC PATHOLOGY, TISSUE PROCESSING, AND DIAGNOSTIC CYTOLOGY

Fixation

• Critical step in tissue preparation.

• Preserves tissue with fixatives.

• Maintains cell/tissue morphology.

• Retains the integrity of biomolecules.

Tissue Handling

• Tissue can be obtained via biopsy, surgical excision, or postmortem.

• Preparation techniques depend on:

  • Structures examined

  • Nature of examined tissues

  • Urgency of investigation

  • Specimen condition (fresh or preserved)

• Fresh tissue examination:

  • Smear (e.g., cancer cervix screening)

  • Frozen section using cryostat (urgent conditions, e.g., during surgery)

Cryostat

• Method involves rapid freezing of fresh tissue using liquid nitrogen or carbon dioxide.

• Sectioning occurs in a cryostat's refrigerated cabinet, maintaining sub-zero temperatures ($-20^\circ C).

Fixatives

• Fixation is a chemical process preserving biological tissues from decay (autolysis or putrefaction).

• Fixation mechanism:

  • Terminates biochemical reactions.

  • Increases mechanical strength and stability of tissues.

Purpose of Fixatives

• Preserve biological material (tissue or cells) close to its natural state.

• Aims of fixation:

  • Prevent postmortem (PM) degeneration.

  • Prevent autolysis (effective against hydrolytic enzymes).

  • Stop bacterial effects.

  • Harden tissues via protein coagulation.

  • Mordanting effect, facilitating tissue staining.

Mechanisms Involved in Fixation

• Additive fixation:

  • Fixative chemical constituent becomes part of the tissue via cross-links.

  • Example: Mercury and Osmium tetroxide

• Non-additive fixation:

  • Fixing agent alters tissue composition without incorporation.

  • Stabilizes tissue by removing bound water from chemical group H-bonds.

  • Example: Alcoholic fixatives

Main Factors Involved in Fixation

• Hydrogen ion concentration:

  • pH of medium should be between 6-8.

• Temperature:

  • Room temperature ($20-25^\circ C$).

  • $40^\circ C$ if using tissue processor.

  • $0-4^\circ C$ for electron microscopy and histochemistry.

• Thickness:

  • 1-2 sq. mm for electron microscopy.

  • 2 sq. cm for light microscopy / 3-5 um.

• Osmolality:

  • Best results with slightly hypertonic solutions (400-450 mosm).

  • Isotonic: 340 mosm.

• Concentration:

  • Formaldehyde - 10% concentration.

  • Glutaraldehyde - 3% for immunoelectron microscopy.

• Duration of fixation:

  • Primary fixation (buffered formalin): 2-6 hrs.

  • Electron microscopy: 3 hrs.

  • Prolonged fixation results in shrinkage and hardening of tissues and inhibits enzyme activity.

Practical Considerations of Fixation

• Speed, rate of tissue penetration, fixative volume

• Speed: Place tissue in fixative ASAP to prevent autolysis.

• Penetration: Formalin diffuses at a rate of 1 mm/hr.

• Volume: Use a 1:20 tissue to fixative ratio for maximum effectiveness.

• Shortening duration of fixation:

  • Heat

  • Agitation

  • Vacuum

  • Microwave

Factors That Affect Fixation

• Retarded by:

  • Thick tissue size

  • Presence of mucus

  • Presence of fat

  • Presence of blood

  • Cold temperature

• Enhanced by:

  • Thin size

  • Agitation

Characteristics of a Good Fixative

1. Kills cells quickly without shrinkage or swelling.

2. Penetrates tissue rapidly.

3. Inhibits bacterial decay and autolysis.

4. Hardens tissue and renders it insensitive to subsequent staining.

5. Allows long-term tissue storage.

6. Simple to prepare and economical to use.

Types of Fixative

• Based on Composition

• According to Action

• Based on Chemical Nature

Types of Fixatives Based on Composition

• Simple fixatives:

  • e.g., Formaldehyde, picric acid, osmium tetroxide

• Compound fixatives:

  • e.g., Bouin's fluid, formol saline, Zenker's fluid

Types of Fixatives According to Action

• Histochemical fixatives:

  Examples:

  • Formaldehyde,

  • glutaraldehyde,

  • vapor fixatives

• Microanatomical fixatives:

  Examples:

  • Bouin's fluid,

  • 10% formalin,

  • Zenker's fluid,

  • formol calcium,

  • Heidenhain's susa,

  • Helly's fluid,

  • Rossman's fluid,

• Cytologic fixatives

  Examples:

  • Champy's fluid,

  • glacial acetic acid,

  • alcohol,

  • formol saline,

  • Carnoy's fluid,

  • Clarke's fluid,

  • Newcomer's fluid,

  • Flemming's fluidure

• Physical agents:

  • Heat, microwaves

• Aldehydes:

  • Formaldehyde, acrolein, glutaraldehyde

• Coagulants:

  • Methyl alcohol, ethyl alcohol, acetic acid

• Oxidizing agents:

  • Osmium tetroxide

• Miscellaneous:

  • Picric acid, mercuric chloride

Aldehyde Fixatives

• Formaldehyde and glutaraldehyde are common.

• They fix tissues by forming cross-links within and between proteins.

Formaldehyde

• Formalin is a solution of formaldehyde, the most common chemical fixative in histopathology.

• Formalin is a 37%-40% solution of formaldehyde gas in water.

• Formaldehyde reacts with functional groups in biomolecules, especially lysine residues in proteins, forming methylene bridges that cross-link and fix tissues.

Principle of Formalin Fixation

• Formalin acts by polymerizing action, forming complexes via methylene bridges between protein molecules.

Formaldehyde Preparation

• 10% formalin is prepared by adding 10 ml of stock formalin to 90 ml of distilled water.

• Turbidity is due to paraformaldehyde formation, removable by filtration.

• Commercial formaldehydes contain 11% to 16% methanol to inhibit paraformaldehyde formation.

Formalin Characteristics

• 8-24 hours fixation time at room temperature.

• 4 hours with agitation.

• 2-3 hours if temperature is increased to 45C.

• Usually buffered to pH 7 with phosphate buffer.

• Unbuffered formalin may cause brown pigments on blood-containing tissues.

Formalin Pigment and Removal

• Formalin pigment is brown granular material formed by formalin in excess blood.

• Removal:

  • Picric acid: Saturated alcoholic solution for 20 min to 2 hrs, then wash with tap water for 10 to 15 min.

  • Kardasewitsch’s method: 70% ethyl alcohol (100 ml) and 20% Ammonia (10-20 ml) for 5 min to 3 hrs, then wash with water.

Glutaraldehyde

• First used in 1962 by Sabatini et al.

• Preserves properties of subcellular structures by EM.

• Renders tissue autofluorescent (less useful for fluorescence microscopy, but can be attenuated by NaBH_4).

• Forms a Schiff's base with amino groups on proteins and polymerizes via Schiff's base catalyzed reactions.

• Forms extensive crosslinks - reacts with the ɛ-amino group of lysine, α-amino group of amino acids - reacts with tyrosine, tryptophan, histidine, phenylalanine and cysteine

• Fixes proteins rapidly but has a slow penetration rate.

• Can cause cells to form membrane blebs.

10% Formal Saline

• Formula:

  • 40% Formalin: 100 ml

  • NaCl: 8.5 Gm

  • Distilled water: 900 ml

• Very safe; maintains color; recommended for CNS material

• Slow and liable for shrinkage during dehydration.

Formol Sublimate

• Formula:

  • 40% Formalin: 100 ml

  • Saturated HgCl_2: 900 ml

• Recommended for routine post-mortem material.

• Cytological details and RBCs are well preserved.

• No hardening/shrinkage.

Formal Calcium

• Recommended for lipid fixation.

• Near neutral pH.

• Formula:

  • 40% Formalin: 10 ml

  • Ca-acetate: 2 Gm

  • Distilled water: 100 ml

• Formalin pigment (acid formaldehyde haematin) is not formed.

Gender's Fluid

• Used for Glycogen.

• Formula:

  • 40% Formalin: 15 mL

  • Picric acid IN 95% alcohol: 80 ml

  • Glacial acetic acid: 05 ml

Advantages of Formalin Fixation

1. Good Tissue Preservation

   • Maintains tissue morphology well, preserving the structural integrity of cells and extracellular components.

2. Prevents Decomposition

   • Effectively halts autolysis and putrefaction.

3. Compatibility with Staining Techniques

   • Works well with most histological stains, especially Hematoxylin and Eosin (H&E).

4. Long-Term Storage

   • Properly fixed tissues can be stored for long periods without significant degradation.

5. Widely Available and Cost-Effective

   • Inexpensive and easy to use, making it practical for routine laboratory use.

6. Penetrates Tissue Well

   • It diffuses through tissue efficiently, allowing relatively uniform fixation (though slower in larger specimens).

Disadvantages of Formalin Fixation

1. Toxicity and Health Risks

   • Toxic, a known irritant, and a potential carcinogen. Proper ventilation and protective equipment are essential.

2. Slow Fixation Process

   • Fixation is not immediate; it can take hours to days depending on tissue size and thickness.

3. Can Mask Antigens

   • Cross-linking can mask certain epitopes, interfering with immunohistochemistry (IHC) unless antigen retrieval techniques are used.

4. Tissue Hardening and Shrinkage

   • Prolonged fixation can lead to hardening, brittleness, or shrinkage of tissues.

5. Potential for Artifact Formation

   • Improper fixation or prolonged storage can cause artifacts like pigment deposition.

6. Environmental Concerns

   • Disposal of formalin waste must follow strict environmental regulations due to its toxicity

Comparison Between Formaldehyde & Glutaraldehyde

Advantages of Glutaraldehyde

• Formation of more cross linkages with better preservation of cellular & fluid proteins

• Resists acid hydrolysis

• Causes less shrinkage than formalin

• More pleasant & less irritant

• Does not corrode metal

• Does not cause dermatitis

Disadvantages of Glutaraldehyde

• Expensive

• Less stable

• Penetrates tissue more slowly than formalin

• Inferior to formalin for PAS satin.

Metallic Fixatives

• Contain heavy metals; used primarily in electron microscopy and sometimes in light microscopy.

• Provide excellent preservation of ultrastructural details.

• Types:

  • Mercuric fixatives

  • Chromate fixatives

  • Lead fixatives

Osmium Tetroxide

• Used in electron microscopy for ultrathin sections.

• Disadvantages:

  • Expensive

  • Slow penetrance

  • Great difficulty in counterstaining

  • Easily reduced by heat, light & dust; store in a cool, dark place in an amber glass bottle.

Mercuric Fixatives

• Mercuric ions combine with acidic (COOH) groups of proteins, forming strong combinations with sulfur (thiol) radicals.

• Types:

  • Zenker's

  • Helly’s Formal (Best for fixation of blood forming or blood containing tissues i.e; spleen or bone marrow

  • Heidenhain’s “Susa “fixative

  • Schaudinn’s sublimated alcohol

  • B5 fixative

Zenker's Fluid

• Recommended for fixing small pieces of liver, spleen, connective tissue fibers, and nuclei.

• Composition:

  • HgCl_2 – 50gm

  • Potassium dichromate – 25gm

  • Sodium sulphate – 10gm

  • Distilled water – 1000ml

  • Add 50ml glacial acetic acid before use (5 ml/dl of stock)

Zenker-formol (Helly's Solution)

• Excellent fixative for pituitary gland, bone marrow, spleen, and liver

• Composition: Zenkers fixative (Mercuric chloride stock solution) + formalin

Heidenhain's Susa Solution

• Recommended for tumor biopsies esp. of the skin.

• Composition: Mercuric chloride + Glacial acetic acid formalin

B-5 Fixative

• Commonly used for Bone marrow samples.

• Composition: mercuric chloride+sodium acetate + distilled water.

Advantages of Mercury Fixatives

• Better staining of nuclei and connective tissue

• Cytoplasmic staining –enhanced with acidic dyes.

• Nuclear chromatin shown in detail

• Preservation of details for photography.

• Best results with metachromatic stain

• B5 fixative is frequently used for bone marrow,spleen, lymph nodes and other hematopoietic tissue

Disadvantages of Mercury Fixatives

• Corrodes the metals

• Lysis of RBC & removes much iron from hemosiderin

• Deteriorates rapidly

• Causes Marked shrinkage

• Reduces the amount of demonstrable glycogen

• Slow penetration

• Tissues become hard & brittle

• Formation of Diffuse black granules in tissues

• Radiopaque: preclude use of x-rays to determine and point of calcification

Precautions-Removal of Black Mercury Granules

• Place sections in 70% alcohol to which scherald solution of iodine is added (0.5ml +100ml alcohol iodine),less for 1 to 2 min to remove merurounchloride deposit.

• 2Hgcl+I2 =Hgcl2+HgI_2

• Rinse in water

• Place in 5% sodium thiosulphate for 1 to 2 min to remove Iodine

• 2 Na2S2O3+I2 = 2 Na I+Na2S4O_6

• Wash in running tap water to remove sodium thiosulphate crystals

Chromate Fixatives

• Chromium salts in H_2O$$ form Cr-O- Cr complexes which have an affinity for the COOH & -OH groups of proteins so that complexes between adjacent protein molecular are formed.

• This leads to disruption of the internal salt linkages of the protein increasing the reactive basic groups & thereby enhancing acidophilic in staining.

Types of Chromate Fixatives

1. Chromic acid

2. Potassium dichromate

3. Regaud's (Muller's fluid)

4. Orth's Fluid

Function of Chromate Fixatives

• Chromic acid - preserves carbohydrates, precipitates proteins

• Potassium dichromate- preserves mitochondria

• Regaud's (Moller's fluid)-Potassium Dichromate +Formalin; used for mitochondria, mitotic figures and golgi bodies.

• Orth's fluid- K dichromate Formalin + Na sulfate; demonstrates rickettsiae, tissue necrosis.

Advantages and Disadvantages of Chromate Fixatives

• Advantages:

  • For demonstration of chromaffin tissues (eg: Adrenal medulla, mitochondria, Golgi apparatus, mitotic figures & RBC’s)

  • Best for preserving phospholipids

• Disadvantages:

  • Prolonged fixation in chromate – Bleach all tissues pigments (melanin)

  • Glycogen preservation is poor

Lead Fixatives

• Lead like other metal fixatives, precipitate proteins. They are used mainly for mucopolysaccharides.

Picric Acid Fixatives

• Picric acid forms protein picrates, some of which are water soluble until treated with alcohol

• Eg: Bouin's fluid

• Brasil’s alcoholic – picro formol fixative

Bouin's Fluid

• For fixation of embryos & pituitary biopsies

• 1.2% aqueous picric acid -75ml

• Formalin – 25ml

• Glacial acetic acid – 5ml

Alcoholic Fixatives

• Rapidly denatures proteins by destroying hydrogen bonds.

• Methyl alcohol

• Ethyl alcohol

• Isopropyl alcohol

• Carnoy's fluid

• Newcomer's fluid

Types of Alcoholic Fixatives and Usage

• Methyl alcohol- excellent for fixing dry and wet smears, blood smears and bone marrow.

• Isopropyl alcohol - for touch preparation.

• Ethyl alcohol- used in histochemistry for enzyme studies.

• Carnoy's fluid- Absolute alcohol + chloroform + glacial acetic acid.

  • Recommended for fixing chromosomes and for urgent biopsies.

  • Considered to be the most rapid fixative.

Acetone

• Recommended for the study of water diffusible enzymes ( ex. lipases & phosphatases)

• For fixing brain tissues for rabies cases.

• Used at Ice cold temperature. -5C - 4C

Heat Fixation

• Ether saline (0.85%) or 10% formal saline is used.

• 20 to 40 ml is heated below the boiling point then the tissue slice (3 to 5mm thick) is placed in hot fluid & heating is continued for 1 min until tissue floats to the surface.

• After this it is cooled quickly in water & mounted on microtome.

Problems Encountered in Fixation

Decalcification

• Optional step in tissue processing.

• Done only if calcium and lime salts are present in the processed tissue (can cause difficulty in cutting).

• Removal of calcium and lime salts.

• Decalcifying agents must be capable of removing calcium without producing considerable tissue destruction.

Types of Decalcifying Agents

1. Acid decalcifying agents

2. Chelating agents ( EDTA)

3. Ion-Exchange resins (Ammonium form of polystyrene resin)

4. Electrical ionization (Electrophoresis)

Different Decalcifying Agents

1. Acid decalcifying agent-

   • Nitric acid

   • Perenyi fluid

   • Phloroglucinol Nitric acid

   • HCl , Von Ebner’s Fluid

   • Formic acid

   • Chromic acid (Flemming’s fluid)

Dehydration

• The process of removing intracellular and extracellular water from the tissue in preparation for wax infiltration.

• Dehydrating agents:

  1. Alcohol (most common)

  2. Acetone (for urgent biopsies)

  3. Dioxane

  4. Cellosolve

  5. Tetrahydrofuran

Using Alcohol (Ethyl - for routine dehydration)

• Tissue is passed in ascending grade/concentration of alcohol

• 70% to 90% to 100% (Absolute alcohol).

Clearing

• Process of removing alcohol from the tissue. Also known as de-alcoholization.

• Renders tissue clear/ghost like.

• Clearing agents:

  1. Xylene- most common

  2. Toluene- substitute for xylene

  3. Benzene- for urgent biopsies

  4. Chloroform- for tough tissus

  5. Cedarwood oil - CNS

  6. Aniline oil- clearing embryos

  7. Clove oil

  8. Carbon- tetrachloride

  9. Tetrahydrofuran

  10. Methyl benzoate

Steps in Tissue Processing (Part 1)

1. Fixation

2. Decalcification (optional)

3. Dehydration

4. Clearing