Histopathology Techniques and Methods of Fixation

What are the two main methods of fixation in histopathology?

Physical methods (heating, microwaving, cryo-preservation) and chemical methods (immersing in fixative solution).

What are the benefits of fixation in histopathology?

1. Allows thin sectioning of tissue by hardening it.

2. Prevents autolysis and inactivates infectious agents.

3. Improves cell avidity for special stains.

What are the steps involved in tissue processing?

1. Fixation

2. Decalcification

3. Dehydration

4. Clearing

5. Impregnation

6. Embedding

7. Sectioning

8. Staining

9. Mounting.

What is the primary goal of fixation?

To preserve the morphologic and chemical integrity of the cell in as life-like manner as possible.

What is the secondary goal of fixation?

To harden and protect the tissue from the trauma of further handling.

What is the recommended fixative to specimen ratio?

20:1 or at least 10:1.

What is the maximum time allowed for specimens to be transferred to fixative?

Less than 1 hour.

What is the significance of the pH level in fixation?

The pH should be between 6-8 for optimal fixation.

What are the routine and electron microscopy temperature requirements for fixation?

Routine: 40℃; Electron Microscopy: 0-4℃.

What is autolysis in the context of histopathology?

Tissue digestion by intracellular enzymes released when organelle membranes rupture.

What is putrefaction?

Bacterial decomposition of tissues.

What are the characteristics of a good fixative?

1. Cheap

2. Stable

3. Safe to handle

4. Inhibits bacterial decomposition and autolysis

5. Allows rapid and even penetration of tissues

6. Hardens tissues for easier cutting

7. Permits application of many staining procedures.

What is the purpose of decalcification in tissue processing?

To remove calcium deposits from tissues to facilitate easier sectioning.

What are the main types of fixatives according to composition?

1. Simple fixatives (e.g., aldehydes)

2. Metallic fixatives (e.g., mercuric chloride, chromate fixatives)

3. Picric acid.

What are the two types of examinations in histopathology?

Biopsy and autopsy.

What is a biopsy?

A procedure to remove a piece of tissue or a sample of cells from a living body for laboratory testing.

What is an autopsy?

A specialized surgical procedure used to determine the cause and manner of death by examining tissues from a dead body.

What is the typical duration for fixation?

24-72 hours.

What is the recommended thickness for tissue blocks in routine fixation?

2 cm² for routine and 1-2 mm² for electron microscopy.

What is H&E staining?

A common staining technique used in histopathology, which stands for Hematoxylin and Eosin.

What is immunohistochemistry?

A technique used to identify specific antigens in tissues using antibodies.

What is the effect of fixatives on soft and friable tissues?

They harden the tissues, making handling and cutting easier.

What is the effect of prolonged decalcification on tissues?

It produces extreme tissue distortion.

What is the primary advantage of using Formalin as a fixative?

It permits relatively good cytologic staining and does not require washing out before dehydration.

What are the disadvantages of using Formalin for decalcification?

The extent of decalcification cannot be measured by a chemical test.

What is the role of Hydrochloric Acid in histopathology?

It is used as a decalcifying agent.

What is the purpose of using Acetone in histopathology?

It is used as a fixative.

What is the function of Osmium Tetroxide in histopathology?

It serves as a fixative.

What are the components of Compound Fixatives?

Examples include Von Ebner's Fluid,

10% Formol Saline,

10% Neutral Buffered Formalin, Heidenhain's Susa,

Formol Sublimate,

Zenker's Solution,

Zenker-Formol,

Bouin's Solution, and

Brasil's Solution.

What are the advantages of using Formic Acid as a fixative?

It permits better nuclear staining than the nitric acid method and is recommended for autopsy materials, bone marrow, cartilage, and tissues studied for research purposes.

What are the disadvantages of using Formic Acid as a fixative?

It is relatively slow and requires neutralization with 5% sodium sulfate.

What are the advantages of Trichloroacetic Acid in histopathology?

It permits good nuclear staining and does not require washing out.

What are the disadvantages of Trichloroacetic Acid?

It is a weak decalcifying agent and is very slow-acting.

What is the significance of Chromic Acid in histopathology?

It acts as both a fixative and a decalcifying agent.

What is the process of decalcification?

It is the removal of calcium ions from a bone or calcified tissue through a histological process.

What is the disadvantage of using Nitric Acid as a decalcifying agent?

The degree of decalcification cannot be measured by routine chemical tests.

What are the advantages of using Citric Acid-Citrate Buffer Solution?

It is rapid in action, produces minimum distortion of tissues, and allows good nuclear staining.

What are the disadvantages of using Chelating Agents in histopathology?

Prolonged use may cause tissue distortion and can seriously damage tissue staining ability.

What is the purpose of dehydration in histopathology?

It is the process of removing water from the specimen and replacing it with dehydrating fluid in preparation for impregnation.

What characteristics should an ideal dehydrating agent have?

It should dehydrate rapidly without producing considerable distortion.

What is the significance of using Neutral EDTA as a chelating agent?

It maintains bone tissue integrity and histological features.

What is the primary disadvantage of using Formol Nitric Acid?

It imparts a yellow color that may impair the staining reaction of the cell.

What is the recommended use for Perenyi's Fluid?

It is recommended for routine purposes.

What is the action of Acetic Acid in histopathology?

It imparts yellowish discoloration to tissues.

What is the role of Nuclear Fixatives in histopathology?

They are recommended for better nuclear staining, especially in autopsy materials.

What are some examples of Cytoplasmic Fixatives?

Examples include Flemming's Fluid without Acetic Acid, Kelly's Fluid, and Formalin with 'post-chroming'.

What are the key advantages of Perenyi's Fluid?

Recommended for routine purposes, good nuclear and cytoplasmic staining, and avoids maceration due to the presence of chromic acid and alcohol.

What are the disadvantages of Perenyi's Fluid?

It can cause shrinkage or distortion of tissues.

What are the characteristics of a good dehydrating agent?

Should dehydrate rapidly, not evaporate quickly, dehydrate fatty tissues, not harden tissues excessively, not remove stains, not be toxic, and not be a fire hazard.

What is the most rapid decalcifying agent mentioned in the notes?

Phloroglucin Nitric Acid.

What is the recommended use for Ethyl Alcohol (Ethanol) in histopathology?

Recommended for urgent cases and routine dehydration of tissues.

What is the boiling point of Ethyl Alcohol?

78.3℃.

What is the recommended procedure for dehydrating delicate tissues using Ethyl Alcohol?

Start with 30% ethyl alcohol and progress to higher concentrations.

What are the advantages of using Xylene as a clearing agent?

It is a colorless clearing agent, rapid dehydrating agent, does not extract methylene blue and other dyes, and is miscible with most embedding resins.

What are the disadvantages of using Xylene?

It evaporates rapidly, is flammable, and some toxicities have been reported.

What are the characteristics of a good clearing agent?

Should be miscible with alcohol and paraffin wax, not produce excessive shrinkage or damage, not evaporate quickly, and make tissues transparent.

What is the recommended use for Methyl Alcohol (Methanol)?

Recommended for blood tissue films and smear preparations.

What is Acetone used for in histopathology?

A rapid-acting dehydrating agent utilized for urgent biopsies.

What is Dioxane's role in histopathology?

Both a dehydrating and clearing agent that produces less shrinkage than ethyl alcohol.

What are the potential toxic effects of Dioxane?

CNS disorders, respiratory depression, abdominal pain, dermatitis, liver diseases, nephrotoxicity, and teratogenic effects.

What is the recommended volume of Xylene relative to tissue?

The volume must be 20 times that of the tissue.

What is the main disadvantage of using Butyl Alcohol (Butanol)?

It is a toxic dehydrating agent.

What is the significance of the term 'clearing' in histopathology?

It refers to the process of removing alcohol or a dehydrating agent from tissue and replacing it with a substance that will dissolve wax for embedding.

What is the primary use of Butyl Alcohol in histopathology?

Recommended for plant and animal micro-techniques.

What should be avoided when using Ethyl Alcohol for dehydration?

Starting with a higher concentration than 70%.

What is the common use of Xylene in histology laboratories?

Used as a clearing agent during tissue processing and as a de-waxing agent during staining.

What is the main advantage of using Dioxane over Ethyl Alcohol?

It produces less shrinkage of the tissues.

What is the recommended environment for using Methyl Alcohol?

It should be used in a well-ventilated room.

What is the primary disadvantage of using Acetone?

It can cause excessive shrinkage and hardening of tissue.

What is a faster dehydrant than ethanol?

A dehydrant that is faster than ethanol is not specified, but it is noted that it has disadvantages such as needing a large volume for dehydration and being toxic.

What are the advantages of Cellosolve as a clearing agent?

Cellosolve is the most rapid clearing agent, suitable for urgent biopsies, clears within 15-30 minutes, makes tissues transparent, is miscible with both absolute alcohol and paraffin, and is cheap.

What are the advantages of Benzene in histopathology?

Benzene may be used in routine paraffin technique, does not excessively harden tissues, and can be used as a dehydrating solution in the staining sequence.

What are the disadvantages of Benzene?

Benzene is expensive and rapidly absorbs water from the air.

What are the properties of Triethyl Phosphate as a clearing agent?

Triethyl Phosphate penetrates and clears tissues rapidly, removes water readily, and produces very little distortion and hardening of tissues.

Why is the use of Triethyl Phosphate discouraged?

Its use is discouraged due to its highly carcinogenic properties.

What are the advantages of Tetrahydrofuran?

Tetrahydrofuran is both a dehydrating and clearing agent, can dissolve fats, is miscible with alcohol, does not make tissues hard and brittle, causes minimum shrinkage, and makes tissues transparent.

What are the disadvantages of Tetrahydrofuran?

Tetrahydrofuran is highly flammable and excessive exposure may cause aplastic anemia.

What are the advantages of Chloroform as a clearing agent?

Chloroform is slower in action than xylene but causes less brittleness, is recommended for routine work, is miscible with absolute alcohol, is suited for tough tissues, and is not flammable.

What are the disadvantages of Chloroform?

Chloroform is relatively toxic to the liver after prolonged inhalation and does not make tissues transparent.

What is the embedding technique in histopathology?

The embedding technique involves placing tissue into a mold containing the embedding medium, which is allowed to solidify.

What is the recommended temperature for immersing tissue in melted paraffin wax?

The recommended temperature is between 5℃ and 10℃ above its melting point.

What is the purpose of orientation in the embedding process?

Orientation is the process by which a tissue is arranged in precise positions in the mold during embedding, on the microtome before cutting, and on the slide before staining.

What is Leuckhart's Embedding Mold?

Leuckhart's Embedding Mold consists of 2 L-shaped strips of heavy brass or metal and is adjustable to give a variety of sizes.

What is a Compound Embedding Unit?

A Compound Embedding Unit is made up of interlocking plates resting on a flat metal base, forming several compartments for embedding multiple specimens.

What are Plastic Embedding Rings?

Plastic Embedding Rings are the most common type of mold used in routine histopathology.

What is the process of impregnation/infiltration in histopathology?

Impregnation/infiltration is the process whereby the clearing agent is completely removed from the tissue and replaced by a medium that fills all tissue cavities and provides firm consistency.

What is a disadvantage of using Xylene as a clearing agent?

Xylene is toxic and narcotic at higher concentrations.

What is the time frame for using Chloroform for embedding?

Chloroform is recommended for routine work within 6-24 hours.

What happens to tissues placed in Chloroform?

Tissues placed in chloroform do not become translucent.

What is the significance of using a clearing agent in histopathology?

A clearing agent is used to remove water from tissues to prepare them for embedding in paraffin.

What is the most common type of mold used in routine histopathology?

Base molds made of plastic or stainless steel.

What is the purpose of embedding molds in histopathology?

To replace tissue with a medium that fills cavities and provides consistency for easier handling and cutting.

What are disposable embedding molds?

Peel-away, thin plastic molds like plastic ice trays and paper boats.

What is the embedding process in histopathology?

The process of placing impregnated tissue into a mold containing a medium that solidifies.

What is microtomy?

The process of trimming and cutting processed tissue into uniformly thin slices for microscopic studies.

What is the basic tool used in microtomy?

Microtome.

What are the characteristics of a good infiltrating agent and embedding medium?

Should be suitable for embedding, easy to handle, and provide good consistency.

What is a Rocking Microtome?

A type of microtome suitable for sectioning and ribboning, invented by Paldwell and Trefall.

What are the advantages of using paraffin wax as an embedding medium?

Allows for thin sections, rapid processing, and indefinite storage of tissue blocks.

What are the disadvantages of using paraffin wax?

Overheating can make specimens brittle, and prolonged infiltration can cause excessive tissue shrinkage.