Histopathology Fixation and Staining

• Fixation is the first and most critical step in histotechnological processes.
• It serves to kill, harden, and preserve fresh tissue for microscopic examination using a fixative.
• Killing stops metabolic processes that alter the tissue state.

Fixatives

• They vary in price and properties
• No single fixative is suitable for all fixation processes.
• The container for a fresh tissue sample should be three-fourths full to meet the fixation needs.
• Inadequate fixation compromises subsequent processes.

Primary Aim

• Preserve cells and surrounding tissues with intact morphology and chemical integrity.
• Preserving cells is vital for correct diagnosis.

Secondary Aim

• Harden and protect tissue from trauma during handling.
• Protect tissues from chemical alterations during histotechnological processes.

Tissue Processes Prevented by Fixation

• Degeneration: Prevents deterioration and function loss in cells, which halts vital processes.
• Decomposition: Prevents the breakdown of organic substances into simpler forms.
• Putrefaction: Prevents protein decomposition, tissue breakdown, and organ liquefaction, which occurs in the decomposition process after death.
• Distortion: Prevents alteration of the original shape of tissue.

Fixative Action

• Fixatives stabilize tissue protein structures, providing mechanical strength to preserve shape and architecture
• Preserves proteins to stabilize and adequately fix tissues.

Preventing Breakdown

• Prevents autolysis by inactivating lysosomal enzymes or chemically stabilizing and insolubilizing tissue components.
• Autolysis, or self-digestion, occurs if tissues aren't preserved with fixatives leading to cellular self-destruction by its own enzymes if tissue isn't preserved.
• Tissues should be preserved immediately upon lab acceptance.

Basic Mechanisms Involved in Fixation

Additive Fixation

• Involves the chemical constituents of the fixative being absorbed and becoming part of the tissue
• Examples include formalin, mercury, and osmium tetroxide.

Non-Additive Fixation

• The fixing agent isn't absorbed, instead it alters the tissue by removing bound water from hydrogen bonds
• Example includes alcoholic fixatives

Coagulant Fixatives

• Create a network for solutions to easily penetrate tissues
• Examples include zinc salts, mercury chloride, picric acid, ethanol, methanol, and acetone.

Non-Coagulant Fixatives

• Create a gel that hinders penetration by subsequent solutions
• Tissues must be thinly cut.
• Tissues are thinly cut to ensure fixatives preserve tissue proteins.
• Thinner tissues allow faster fixative penetration due to the tough outer surface.

Main Factors Involved in Fixation

• Hydrogen Ion Concentration: Maintain a pH between 6.0 and 8.0.

Temperature

• Fixation commonly occurs at room temperature (20-24°C)
• For electron microscopy, the fixation process takes place between 0-4°C.
• Heat Fixation: rapid fixation used in bacteriology and blood films.
• Formalin heated at 60°C enables rapid fixation of urgent biopsy samples.
• Formalin heated at 100°C is specifically used when dealing with tissues affected by tuberculosis.

Thickness of Section

• Plays a role in the duration of the fixation process

Thickness Guidelines

• Electron microscopy: 1 to 2 mm^2
• Light microscopy: 2 cm^2, not exceeding 0.4 cm
• Brain tissue is typically suspended whole in 10% buffered formalin for 2-3 weeks for proper hardening and fixation

Osmolality

• Using a slightly hypertonic solution (400-450 mOsm) is best.
• Sucrose is added to osmium tetroxide fixatives.
• Hypertonic solutions cause cell shrinkage while hypotonic solutions cause cell swelling

Concentration

• Formaldehyde: 10% solution

Concentration Guidelines

• Glutaraldehyde: 3% solution, 0.25% is ideal for immunoelectron microscopy

Duration of Fixation

• Formalin should be applied for 2-6 hours on the day the specimen is obtained.
• Insufficient duration results in inadequate fixation.
• Prolonged formalin fixation shrinks and hardens tissue, inhibiting enzyme activity and immunologic reactions.
• For electron microscopy, tissues are fixed for 3 hours then placed in a holding buffer for subsequent processes.
• Tissues should be immersed in fixative within 60 minutes of blood supply interruption.
• Using 10% NBF (Neutral-Buffered Formalin): Requires a minimum of 6 hours up to a maximum of 48 hours.

Practical Considerations of Fixation

• Speed: Tissues must be fixed immediately upon arrival in the lab to avert autolysis and putrefaction.
• Commence as soon as it is removed from the body

Penetration

• The fixing agent must penetrate tissue at approximately 1 mm per hour.
• Fixation time differs with each tissue type.
• Tougher tissues require longer penetration times.

Volume

• Ratio must be 10-25 times greater than the tissue volume to be fixed to adequately fix the entire specimen
• For max effectiveness, the fixative must be 20 times greater than the tissue volume
• 1:20 is the appropriate tissue to fixative ratio.

Duration

• Fibrous organs (uterus or intestinal tract) need longer fixation than small/ loosely textured-tissues
• Fixation time can be reduced via heat, vacuum, agitation, or microwave

Methods of Fixation

• Chemical Fixation involves chemicals.
• Vapor Fixation helps to retain soluble materials in situ like materials retain soluble substances
• Heat Fixation involves thermal coagulation of proteins facilitating rapid diagnosis.
• It is Commonly used for bacteriologic smears.
• Microwave Irradiation: uses non-ionizing radiation for fixation
• Fixation results from heat and rapid molecular movement with electromagnetic flux
• Ultrasound Fixation: Uses a high frequency, high intensity, ultrasonic apparatus giving better results and shortens fixation time.

General Effects of Fixatives

• Soft and friable tissues are hardened, preserving life-like manner.
• Cells are made resistant to damage and distortion..
• Bacterial Decomposition is inhibited.
• Increase optical differentiation of cells and tissue components.
• Serves as mordants or accentuators and may inhibit certain dyes.

Characteristics of a Good Fixative

• Should be cheap, stable, safe to handle, and able to rapidly kill the cells
• While there is no known ideal fixative the most common fixative is still 10% Neutral-buffered Formalin.

Types of Fixative

According to Composition

• Simple fixatives are made up of only one component.
• Compound fixatives are made up of two or more substances

According to Action

• Microanatomical fixatives permit the general microscopic study of tissue structures without altering structural pattern, and normal intercellular relationship of the tissues in question
• Cytological fixatives preserve more specific parts, and particular microscopic elements of the cell itself
• Nuclear fixatives preserve the nuclear structures.
• They usually contain glacial acetic acid due to its high affinity to nuclear chromatin with a pH of 4.6 or less
• Cytoplasmic fixatives preserve cytoplasmic structures.
• They must never contain glacial acetic acid which destroys mitochondria and Golgi bodies with a pH must be more than 4.6.
• Histochemical fixatives preserve the chemical constituents of the cell.

Lipid Fixation

• Mercuric chloride and potassium dichromate are effective in preserving lipids in cryostat sections.
• Baker's formol-calcium preserves phospholipids
• Imidazole osmium tetroxide improves the ultrastructural demonstration of lipids.
• Digitonin Cholesterol fixation, preserves cholesterol in the Tissue

Carbohydrate Fixation

• Alcoholic fixatives Recommended for glycogen fixation.

Protein Fixation

• Neutral buffered formol saline or formaldehyde vapor is most commonly used and are fixatives for acid histochemistry
• Karnovsky’s paraformaldehyde- glutaraldehyde solution is the best known fixative for electron cytochemistry

Acrolein

• Mixture of glutaraldehyde or formaldehyde. Rapidly preserves the morphology and enzyme activity at low concentration and is also useful for immersion fixation of surgical biopsies

Aldehyde Fixatives

• These fixatives produce a satisfactory result for routine paraffin sections, such as light microscopic studies.

Formaldehyde

• It Is produced by oxidation of methyl alcohol.
• Recommendation: Use 10% formalin.
• A pure stock solution(40%) over hardens the outer layer of tissue which in trun, affects staining aversely.
• Recommendation: buffered to a pH of 7.0 with phosphate buffer.
• Fixation Time: 24 hours.
• Is also recommended, for colored tissue photography.
• It is a tolerant fixative that is used for mailing specimens.
• By not buffered, pH is 4 and has little effect but produces creates a dark pigment which can obscure cellular details.
• Formaldehyde when not buffered, creates a precipitate.

Methanol

• It is a preservative added to formaldehyde to prevent its decomposition to formic acid or precipitation to paraformaldehyde.
• It denatures protein, making formailn unsuitable for electron microscopy

Precautions

• Induce precipitation at very low temperatures
• Bleaching of tissues may be prevented by changing the fluid 3 months
• brown or black crystalline precipitate should be treated with alcoholic acid.
• Magnesium Carbonate is added to neutralize in order to neutralize acid reactions

###10% Formol-Saline

• This is made up of formaldehyde diluted to 10% with with sodium chloride
• Is commonly used for central nervous system tissues
• And it ideal for tissue staining and minimizes shrinkage during alcohol dehydration

10% Neutral Buffered

• Formaldehyde: Used for preservation and post Mortem specimens
• It prevents precipitation of the acid and produces a tissue that is clear

Formol-Corrosive

• Recommended for routine tissues.
• Inhibits the growth of organ tissues and penetrates small pieces rapidly.
• There is no need to “wash-out” the tissue, after applying the fixative

Alcoholic Formalin

• Commonly used for post fixation
• Fixation is faster but it also produces gross gross hardening of tissues.

Glutaraldehyde

• Used in small tissue fragments.
• The recommendation used in the microscopic is buffered to perserve the stain.

Metallic Fixatives

• Mercuric Chloride: The recommendation of renal tissues is to to extract the muscles.
• Used in acid solutions.

Zenker's Fluid

• Used acid based for spleen and conective tissues
• Made of mercuric chloride

Heidendhains Susa Solution

• The time must last between 3-12 hrs, this can be removed through and fixation tissues

Chromate Fixitives

• Used to presreve carbs, and should a string reducing agent

This lesson covers methods for rapid diagnosis using thermal coagulation of proteins, fixatives, and staining techniques. It contains topics such as vapor fixation, osmium tetroxide, and Flemming's solution.