Fixation Histopathological Specimen

UNIT III

Fixation (Histological Specimens and Cytological Specimens)

• 3.1.1 Classification of fixatives

• 3.1.2 Composition of various fixatives

• 3.1.3 Advantages and Disadvantages

• 3.2 Processing of Tissue (by Paraffin Technique)

• 3.2.1 Dehydration

• 3.2.2 Clearing/Dealcoholization

• 3.2.3 Infiltration and Impregnation

• 3.2.4 Paraffin embedding

• 3.2.5 Mountants

• 3.2.6 Various types of mounting media (aqueous, resinous), Advantages and Disadvantages

Classification of fixative:

Fixatives for histological specimens and cytological specimens are classified into two groups based on chemical composition and their mode of action. These are simple fixatives and compound fixatives.

1. Simple fixatives

   • · Aldehyde

   • · Formalin

   • · Glutaraldehyde

   • · Alcohol: Ethanol and Methanol

   • · Mercurials: Mercuric chloride,

   • · Organic solvent: Acetone, Xylene

   2. Compound Fixatives

   • · Bouin’s solution

   • · Zenker’s fixative

   • · Carnoy’s fixative

   • · B5 fixative:

3.1.2 Composition of various fixatives

1. Simple fixatives: Simple fixatives used for histological specimens and cytological specimens consist of one ingredient. Simple fixatives are further sub-classified into the following groups based on their chemical composition

   • · Aldehydes: Formaldehyde and Glutaraldehyde.

   • Formalin is the most commonly used fixative in histopathology. 10% to 40% formaldehyde solution in water is formalin. Commercial formalin is a Formaldehyde solution of 40% v/v in water. A neutral buffered Formaldehyde solution of 10% v/v in water is most commonly used in medical laboratory technology as a fixative. It is considered as a universal fixative. Formalin preserves tissue structure, cellular morphology, and proteins very effectively. Formalin develops cell proteins cross-linking, cell stabilization, and preservation.

     Composition of 10% Neutral buffered Formalin

     • Sodium phosphate, monobasic 4 gm

     • Sodium phosphate, dibasic 6.5 gm

     • Formaldehyde 40% v/v: 100 mL

     • Distilled Water 900 mL.

     Formalin is a primary fixative, while others are alternate fixative under the simple fixative category.

   • Glutaraldehyde 70% w/w is an effective fixative. But it penetrates tissues slowly. It better preserves ultra-cellular structure and makes it suitable for electron microscopy.

   • · Alcohol: Ethanol, methanol

     • Ethanol 50 to 60% v/v is used as a fixative. It is a commonly used dehydrating agent in histopathology. It acts as a fixative for lipid-rich certain tissues.

     • Methanol above 80% v/v is used as a preservative in blood smears and cytological preparation.

   • · Mercurials: Mercuric chloride

   • Mercuric chloride is used as an ingredient in compound fixatives such as B5 fixative and Zanker’s fixative. Mercuric chloride alone is rarely used as a fixative due to its toxic properties. It is mainly used to preserve nucleic acid and in some special staining techniques.

   • · Organic solvents: Acetone, xylene,

     • Acetone is a commonly used fixative for frozen sections. It rapidly dehydrates tissue to preserve antigenicity.

     • Xylene is used as a fixative but it is a clearing agent to remove alcohol from tissue.

2. Compound fixatives: Compound fixatives consist of two or more ingredients. The following is used as a compound fixative for histological specimens and cytological specimens.

   • · Bouin’s solution: It is a mixture of picric acid, formaldehyde, and acetic acid. It is used to preserve delicate structures such as testicular tissues.

     • Saturated picric acid 1500 mL

     • Formaldehyde 500 mL

     • Glacial Acetic Acid 100 mL

   • · Zenker’s fixative: It is a mixture of formaldehyde, mercuric chloride, and potassium dichromate. It is commonly used for lymph node and liver biopsies.

     • Mercuric chloride 50 gm

     • Potassium dichromate 25 gm

     • Sodium sulphate 10 gm

     • Distilled water 1000 mL

   • · Carnoy’s fixative: It is a mixture of ethanol, chloroform, and acetic acid. It is used to preserve chromosomes and nuclear structure.

     • Ethanol 60 mL

     • Chloroform 30 mL

     • Glacial acetic acid 10 mL

     • Ferric chloride 1 gm

   • · B5 fixative: It is a mixture of formaldehyde and mercuric chloride. It is used for bone marrow biopsies and lymphoid tissues.

     • Mercuric chloride 12 gm

     • Sodium acetate 2.5 gm

     • Distilled water 200 mL

Advantages and disadvantages of fixatives

1. Formalin

          Advantages of Formalin as fixative·

• Widely available and cost-effective fixative

• · Easy to use.

• · Rapid tissue penetration,

• · No tissue hardening,

• · Excellent tissue preservation,

• · Minimises autolysis and putrefaction,

• · Suitable for long-term tissue storage,

• · Compatibility with various histopathological processes,

• · Compatible with staining techniques,

• · Safe fixative

       Disadvantages of Formalin as fixative·

• Tissue shrinkage and distortion of cellular features,

• · Abnormal changes in tissue·

• Cross-linking of protein and nucleic acid makes tissue less accessible to stain

• · Loss of enzymatic activity in tissues,

• · Frequent over-fixation and under-fixation

• · Large tissues require a long fixation time

• · Formalin vapor develops allergic reactions like irritation to the eyes, nose, respiratory tract, skin, etc.

      2. Alcohol

        Advantages of Alcohol as a fixative

• · Rapid and quick fixation

• · Low tissue shrinkage

• · Better preservation for lipids and pigments

• · Compatible with molecular analysis

• · No health risks like allergies,

      Disadvantages of Alcohol as a fixative

• · Not proper preservation of nuclear and cytoplasmic structures

• · Not suitable for some immunohistochemical markers,

• · Evaporation of alcohol during the fixation process.

• · Not suitable for several tissues,

• · Costly and ethanol is not available readily.

      3. Murcurials: Mercuric chloride

      Advantages of Mercuric chloride as a fixative

• · Rapid fixation

• · Good for nuclear and cytoplasmic details

• · Minimal tissue shrinkage,

• · Compatible with certain stains.

   Disadvantages of Mercuric chloride as a fixative

• · Extremely toxic

• · Risk of mercury poisoning

• · Environmental hazard

• · Tissue hardening

• · Poor preservation

• · Limited compatibility

• · Tissue discoloration

      4. Acetone

       Advantages of Acetone as a fixative

• · Economical and easy availability

• · Rapid Fixation,

• · No chemical cross-linking

• · Minimum tissue shrinkage

• · Preserves certain antigens

     Disadvantages of Acetone as fixative

• · Limited tissue penetration

• · Cellular distortion

• · Poor lipid preservation

• · Incompatible with certain stains

• · Potentially harmful chemical

      5. Bouin’s solution:

       Advantages of Bouin’s solution as a fixative

• · Rapid fixation

• · Wide acceptability,

• · Clear nuclear content visibility,

• · Excellent tissue preservation

• · Good compatibility with stains

        Disadvantages of Bouin’s solution as a fixative

• · Tissue shrinkage and distortion

• · Strong fumes, unpleasant odor, and Hazardous chemicals

• · Incompatible with certain staining techniques

• · Not suitable for long-term storage of tissues

• · Variable results.

     6. Zenker’s fixative

      Advantages of Zenker’s fixative

• · Rapid fixation

• · Excellent tissue preservation

• · Clear nuclear content visibility

• · Relatively economical

   Disadvantages of Zenker’s fixative

• · Tissue shrinkage and distortion

• · Limited antigen preservation

• · Toxic and hazardous chemicals

• · Difficult disposal

• · Not environmentally friendly

   7. Carnoy’s fixative

        Advantages of Carnoy’s fixative

• · Rapid fixation

• · Excellent nuclear and cytoplasmic preservation

• · Reduced tissue shrinkage and reduced tissue distortion

• · Compatible with various stain

        Disadvantages of Carnoy’s fixative

• · Brittle tissue section

• · Loss of certain antigen

• · Difficult to handle

• · Limited penetration

• · Not suitable for all tissues.

   8. B5 fixative

       Advantages of B5 a fixative

• · Good preservation of tissue structure,

• · Chemical Fixation,

• · Widely available,

• · Suitable for long-term storage of tissues,

• · Compatible with staining techniques,

• Disadvantages of B5 fixative

• · Time-consuming fixation process,

• · Interfere with molecular studies,

• · Improper fixation

• · Formaldehyde exposure to laboratory technicians,

Dr Pramila Singh

Processing of Tissue (by Paraffin Technique)

Processing of tissues using paraffin is a standard method to prepare tissue samples for microscopic examination. This process requires several steps to prepare the tissue. These steps are fixation, dehydration, clearing, Infiltration, impregnation, paraffin embedding, sectioning, staining, and cover-slipping.

• Fixation: It is the first step to process a tissue specimen obtained through biopsy or surgery. Immerse tissue into a fixative solution immediately after its collection. It prevents the degradation of tissue structure by stabilizing the protein and cellular structures, preventing autolysis (self-degradation) and putrefaction.

• Dehydration: It is the second step. Dehydration is the removal of water molecules from the tissue specimen. It is carried out by passing the tissue specimen through a series of alcohols of various strengths such as Ethanol 70%, Ethanol 80%, Ethanol 95%, and absolute ethanol (Ethanol 100%). There will be progressive dehydration. Paraffin does not embed moist tissues. Thus dehydration of tissue specimens is carried out to facilitate the embedding step.

• Clearing/ decolorization: It is the third step. Dehydrated tissues contain alcohol. Alcohol is immiscible with paraffin. Alcohol is washed out from tissues by using clearing agents such as xylene or other organic solvents. The clearing agent makes tissues transparent. It also makes tissue specimens suitable for the entry of paraffin wax into tissues.

• Infiltration and Impregnation: It is the fourth step. Entry of paraffin wax into tissue specimens is called infiltration of paraffin wax. Place dehydrated and clear tissue specimens into molten paraffin wax. Apply a vacuum to remove air bubbles trapped within the tissue specimen. Paraffin wax entry into tissue replaces the clearing agent completely from the tissue specimen. Infiltration of paraffin provides support to tissue structure. It makes tissues suitable for tissue section cutting.

• Paraffin Embedding: Embedding step is carried out after complete infiltration and impregnation of paraffin into tissues. Place impregnated tissues into molds. Pour more fresh molten wax around it. The orientation of tissue in the mold should be appropriate. This ensures proper section cutting of the tissue. Allow paraffin wax to solidify. It will form a solid block. The block is ready for section cutting.

• Sectioning: Use microtome to cut thin sections of paraffin-embedded tissue block. Adjust the thickness of the section cutting around 4 to 5 micrometers. A thin section is mounted on the clean sterile glass slide.

• Mounting: A thin section of tissue shall float on the surface of the water bath. Carefully pick and place the section of tissues onto a clean sterile glass slide. Dry it and ensure the adhesion of the thin section onto the surface of the glass slide.

• Staining: It is the final step of tissue processing. Staining highlights the cellular structure and cellular components. Hematoxylin and eosin stain (H & E stain) is a commonly used stain in histopathology. It provides excellent contrast between cell nuclei (blue) and cytoplasm (pink).

• Cover slipping: Place cover slip over the stained tissue section using the proper mounting medium. Cover-slip protects tissue section during microscopic examination. It also ensures the tissues remain in a fix position during microscopic examination.

Various types of mounting media (aqueous, resinous):

Mounting media secure cover slip over the thin section of stained tissues on a glass slide. Mounting media enhances the visibility of stained tissue under a microscope, protects tissues during microscopic examination, and fixes the position of stained tissue on a slide during microscopic examination. There are three types of mounting media. These are aqueous mounting media, resinous (organic) mounting media, and synthetic mounting media.

1. Aqueous mounting media: They are water-based solutions suitable for water-soluble dyes (stains) such as Hematoxylin and eosin stains (H & E stain). They are suitable for a simple and quick mounting process. Examples: Aqueous mounting medium and Glycerol-Gelatin mounting medium.

2. Aqueous mounting medium is a clear, water-based medium. It does not require dehydration of the tissue sections. The tissue section can be directly mounted from the water bath.

3. Glycerol-Gelatin mounting medium: It contains gelatine and glycerine. It helps to preserve the tissue section and enhance tissue transparency. It is used for temporary mounts. It can be easily removed if re-staining is required or any further processing is required.

4. Resinous (organic) mounting media: They are used for tissue sections stained with alcohol or xylene-based stains (dyes). They require dehydration and clearing of tissue sections. Examples: DPX Mounting medium (Distyrene Plasticizer, xylene), Entellan, Canada Balsam

5. DPX Mounting medium (Distyrene Plasticizer, xylene): It is a synthetic resinous mounting medium. Its main components are Distyrene, Plasticizer, and xylene. Distyrene is a synthetic resin. Dieterene forms a hard and durable cover over the tissue section. It protects tissue sections from physical damage and environmental factors. It remains intact during storage and handling due to the hard nature of Distyrene. Plasticizer improves flexibility and adherence of resin to the slide. Plasticizer prevents the formation of bubbles or gaps between tissue section and cover slip. Xylene is a solvent to dissolves Distyrene and plasticizer to form a clear solution. Xylene makes DPX mounting medium compatible with most of the histological stain.

6. Synthetic Mounting Meida: Permount, Vectashield.

    Advantages of Aqueous mounting media:

• 1. Easy to use,

• 2. Compatible with water-soluble stain

• 3. Easily washed out from tissue section,

• 4. Minimum tissue shrinkage and tissue alteration

    Disadvantages of Aqueous mounting media:

• 1. Swelling of mounting media and cover-slip

• 2. Not suitable for long-term preservation of tissue section

• 3. Not suitable for stains soluble in organic solvent.

     Advantages of Resinous mounting media

• 1. Long term preservation

• 2. Exceptional transparency

• 3. Durability

       Disadvantages of Resinous mounting media

• 1. Inflammable and toxic

• 2. Xylene of resinous mounting media is a volatile organic solvent that is hazardous.

Dr Pramila Singh