Fixation (Part 1)- MIDTERMS L8

FIXATION

• Classically defined as killing, penetration and hardening of tissues.

• Currently defined as the alteration of tissues by stabilizing protein so that the tissues become resistant to further changes

• No process of histotechnology is more critical to slide preparation than fixation.

PRIMARY GOAL OF FIXATION

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

SECONDARY GOAL OF FIXATION

Harden and protect tissue from trauma of further handling, so that it is easier to cut during gross examination

STABILIZATION OF PROTEINS

Most important reaction in maintaining the morphology of the tissue in fixation

MOST COMMON ERROR IN HISTOTECHNOLOGY

Insufficient ratio of tissue volume to fixative volume

ACTIONS OF FIXATIVE

• Preserve the tissue.

• Prevent breakdown of cellular elements.

• Coagulate or precipitate protoplasmic substances

SUICIDE SAC

(Lysosomes)- AUTOLYSIS AND PUTREFACTION

MAIN FACTORS INVOLVED IN FIXATION

1. pH (Hydrogen Ion Concentration)

2. Temperature

3. Thickness of section

4. Osmolality

5. Concentration

6. Duration of fixation

7. Volume

8. Time Interval

pH

• RANGE: pH 6-8

• OPTIMAL: pH 7

TEMPERATURE

• Most tissue processes: 40*C

• EM and Histochemistry: 0-4* C (Mast Cell: room temperature)

• General: RT

• Rapid Formalin: 60*C

THICKNESS

EM: 1-2 mm²

LIGHT MICROSOPY: 2cm² wide, and 0.4 cm thick (4mm)

BRAIN

• Should be fixed before cutting- 10% buffered formalin for 2-3 weeks

OSMOLALITY

Slightly hypertonic solution: 400-450 mOsm

Isotonic solution: 340 mOsm

SUCROSE

Is added to osmium tetroxide fixatives for EM

CONCENTRATION

• Lowest level possible

• FORMALIN: 10%

• GLUTARALDEHYDE: 30%/ 0.25% (EM)

DURATION OF FIXATION

• Fibrous organs have longer fixation than biopsies or scrapings.

• Can fasten thru heat, vacuum, microwave, and agitation

VOLUME

10-20 times greater than the tissue

TIME INTERVAL

• Time between the tissue has been removed from the patient until it placed on a fixative.

• The longer the blood supply is interrupted, the poorer the quality of the tissue

PRACTICAL CONSIDERATIONS OF FIXATION

1. Speed- <1hr. after surgery

2. Rate of Penetration- Formalin diffuses 1 mm/ hr.

3. Volume- 10-25x ratio, best is 20

4. Duration of Fixation

EFFECTS OF FIXATIVE IN GENERAL

1. Harden soft friable tissues and make the handling and cutting of sections easier.

2. They make cells resistant to damage and distortion by hypotonic (swell) and hypertonic (shrink) solutions used.

3. Inhibit bacterial decomposition.

4. Increase optical differentiation of cell and tissue components.

5. They act as mordants or accentuators to promote and hasten staining.

6. Reduce risk of infections during handling and actual processing of tissues.

CHARACTERISTICS OF GOOD FIXATIVES

• It must be cheap, stable, and safe to handle.

• It must kill cells quickly; It must inhibit bacterial decomposition and autolysis.

• It must produce minimum shrinkage of tissues.

• It must permit rapid and even penetration of tissues; It must harden tissues.

• It must make cellular components insoluble by hypotonic solution and render them insensitive to subsequent processing.

• It must permit application of many staining procedures.

• It must be isotonic causing minimal physical and chemical alteration of the cells and their constituents.

MISCELLANEOUS CONSIDERATION

• Tissue selected for sectioning should be thin enough to allow penetration by fixative within a reasonable amount of time

• Most tissue can be cut and trimmed without prior fixation, EXCEPT for the BRAIN which is generally soft when unfixed.

• Refrigerator is used to slow down decomposition if the tissue needs to be photographed and cannot be fixed immediately

FACTORS THAT AFFECT FIXATION OF TISSUES

• Factors that ENHANCE (ACCELERATES) the fixation

• Factors that RETARD (SLOWS) the fixation

FACTORS THAT ENHANCE (ACCELERATES) THE FIXATION

1. Size and thickness of tissue (smaller and thinner tissues)

2. Agitation

3. Moderate heat: 35-36*C

FACTORS THAT RETARD (SLOWS) THE FIXATION

1. Size and thickness of the tissue (large)

2. Presence of mucus

3. Presence of fat

4. Presence of blood

5. Cold temperature

TWO BASIC MECHANISMS INVOLVED IN FIXATION

1. Additive fixation

2. Non- additive fixation

ADDITIVE FIXATION

• Chemical constituent of the fixative is taken in and becomes a part of the tissue

• For cross links to stabilize protein

EXAMPLES OF ADDITIVE FIXATIVE

• Formalin

• Mercury

• Osmium tetroxide

NON- ADDITIVE FIXATION

• Fixing agent is not taken into the tissue. But it alters the tissue composition thus, stabilizing the tissue which makes them unsuitable for bacterial decomposition.

• Removal of water→ new cross links will be established→ prevents autolysis and putrefaction

EXAMPLE: alcoholic fixatives

TYPES OF FIXATIVE ACCORDING TO COMPOSITION

1. Simple fixative

2. Compound fixative

SIMPLE FIXATIVE

Made up of only one component substance.

COMPOUND FIXATIVE

Made up of two or more fixatives.

TYPES OF FIXATIVES ACCORING TO ACTION

1. Microanatomical Fixatives

2. Cytological Fixatives

3. Histochemical Fixatives

MICROANATOMICAL FIXATIVES

• Are those that permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissue in question.

• It must never contain osmium tetroxide because it inhibits hematoxylin.

CYTOLOGICAL FIXATIVES

Are those that preserve specific parts and particular microscopic elements of the cell

1. Nuclear Fixatives

2. Cytoplasmic Fixatives

NUCLEAR FIXATIVES

• Preserve the nuclear structures

• Contain glacial acetic acid as their primary component due to its affinity for nuclear chromatin

• pH 4.6 or less

CYTOPLASMIC FIXATIVES

• Preserve the cytoplasmic structures

• Must NEVER contain glacial acetic acid which destroys the mitochondria and Golgi bodies

• pH of more than 4.6

HISTOCHEMICAL FIXATIVES

Are those that preserve the chemical constituents of cells and tissues.

EXAMPLES OF MICROANATOMICAL FIXATIVES

• ALDEHYDE FIXATIVES

  • 10% Formol Saline

  • 10% Neutral Buffered Formalin

  • Formol Sublimate (Formol Corrosive)
    

• PICRIC ACID

  • Bouin’s solution

  • Brasil’s solution

• MERCURIC CHLORIDE

  • Heidenhain’s Susa

  • Zenker’s Solution

  • Zenker-Formol (Helly’s)

EXAMPLES OF NUCLEAR FIXATIVES

1. Bouin’s fluid

2. Flemmings with acetic acid

3. Newcomer’s

4. Carnoy’s fluid

5. Heidenhain’s Susa

“BENCH“

CYTOPLASMIC FIXATIVES

1. Helly’s

2. Orth’s fluid

3. Regaud’s

4. Flemming’s fluid WITHOUT Acetic acid

5. Formalin with Post Chroming

“HORFF“

HISTOCHEMICAL FIXATIVES

• Acetone

• Absolute Ethyl Alcohol

• 10% Formol Saline

• Newcomer’s fluid

BOTH MICROANATOMIC AND NUCLEAR FIXATIVE

• Heidenhain’s

• Bouin’s

BOTH HISTOCHEMICAL AND NUCLEAR FIXATIVE

Newcomer’s

BOTH HISTOCHEMICAL AND MICROANATOMICAL FIXATIVE

• 10% Formol saline

BOTH MICROANATOMIC AND CYTOPLASMIC FIXATIVE

Helly’s (Zenker- Formol)

SECONDARY FIXATION

• A process wherein an already fixed tissue specimen is placed in a second fixative.

• Before dehydration or before staining

SECONDARY FIXATION

1. To facilitate and improve the demonstration of a particular substance.

2. To make special staining techniques possible (secondary fixative acting as mordant).

3. To ensure complete and further hardening and preservation of tissues

POST CHROMATIZATION

A form of secondary fixation whereby a primarily fixed tissue is in a 2.5 to 3% Potassium Dichromate solution for 24 hours to act as mordant for better staining effects and to aid in cytologic preservation of tissues

WASHING OUT

Process of removing excess fixative from tissue after fixation to improve staining, and to remove artifacts from the tissues.

SOLUTION USED FOR WASHING OUT

1. Tap water

2. 50 to 70% Alcohol

3. Alcoholic Iodine

TAP WATER

1. Excess Chromates fixed in Helly’s, Zenker’s, and Flemming’s Solutions.

2. Formalin

3. Osmic Acid

50 TO 70% ALCOHOL

Picric Acid (Bouin’s)

ALCOHOLIC IODIDE

Mercuric fixative

DEZENKERIZATION

• Mercuric deposits may be removed by immersing tissues in alcoholic iodine solution prior to staining through a process known as dezenkerization. Chemically, this is done by the oxidation with sodium to mercuric iodide, which can be subsequently removed by treatment with sodium thiosulfate.

• Mercurial fixatives and reagents used in dezenkerization MUST NOT go through drain disposal.

PHYSICAL METHOD OF FIXATION

1. Heat Fixation

2. Microwave Fixation

3. Freeze-Drying and Freeze Substitution

HEAT FIXATION

• Accelerate other forms of fixation as well as the steps in tissue processing.

• Usually employed for frozen tissues sections and preparation of bacteriologic smears

MICROWAVE FIXATION

• Works as a physical agent. Agitation to increase the movement of molecules and accelerate fixation.

• Allows light microscopic techniques.

• Microwave treated tissue (at 50%), post fixed in osmium tetroxide, gives satisfactory results for electron microscopy

FREEZE- DRYING AND FREEZE SUBSTITUTION

• Freeze drying is a special way of preserving tissues by rapid freezing (quenching -160*C to -180*C. Higher temperature (sublimation -30*C to -40*C)

• Freeze substitution is similar to freeze drying, the only variation is that the frozen tissues instead of being subjected to dehydration in an expensive vacuum drying apparatus – is fixed in Rossman’s formula or in 1% acetone and dehydrated in absolute alcohol.