LECTURE 2: Fixation (Part 1)

HISTOTECHNOLOGY

Is the art and science performed to produce a tissue section of good quality that will enable the pathologist to diagnose the presence or absence of a disease.

FIXATION

The first and most critical step in histotechnology

FIXATION

A process that preserves tissues from decay, thereby preventing autolysis or putrefaction.

fixing or preserving a fresh tissue for examination

The first and most critical step in histotechnology involves?

FIXATION

A process adopted to kill, harden and preserve materials for microscopic study by means of a fixative.

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

The primary aim of fixation is to?

harden and protect the tissue from the trauma of further handling

The secondary aim of fixation is to?

1. Degeneration

2. Decomposition

3. Putrefaction

4. Distortion

TISSUE CELLULAR PROCESSES PREVENTED BY FIXATION:

DEGENERATION

The state or process of being or becoming degenerate; decline or deterioration.

DEGENERATION

Deterioration and loss of function in the cells of a tissue or organ.

MACULAR DEGENERATION

Degeneration of the central part of the retina.

DECOMPOSITION

Is the process by which organic substances are broken down into a much simpler form of matter.

PUTREFACTION

One of the 7 stages in the decomposition of the body after death

PUTREFACTION

Decomposition of proteins in a process that results in the eventual breakdown of cohesion between tissues and the liquefaction of most organs.

DISTORTION

Is the alteration of the original shape (or other characteristic) of a tissue.

proteins

To maintain tissue morphology, fixation stabilizes the ____ in the tissue

insoluble

Soluble proteins are fixed to structural proteins and thus rendered (soluble/insoluble).

FIXATION

This gives its mechanical strength that preserves the shape and even the architectural pattern of the tissue.

AUTOLYSIS

Fixation prevents “____” by inactivating the lysosomal enzymes or by chemically altering, stabilizing and making the tissue components insoluble.

AUTOLYSIS

Is more commonly known as “self digestion”.

self digestion

Autolysis is more commonly known as?

AUTOLYSIS

It refers to the destruction of a cell through the action of its own enzymes.

1. Additive Fixation

2. Non-additive Fixation

3. Coagulant Fixatives

4. Non-coagulant Fixatives

BASIC MECHANISMS INVOLVED IN FIXATION:

ADDITIVE FIXATION

Whereby the chemical constituent of the fixative is taken in and becomes part of the tissue.

ADDITIVE FIXATION

Identify the Basic Mechanism involved in Fixation:

Examples:

1. Formalin

2. Mercury

3. Osmium tetroxide

NON-ADDITIVE FIXATION

Whereby the fixing agent is NOT taken in, but changes the tissue composition and stabilizes the tissue by removing the bound water attached by the hydrogen bonds.

NON-ADDITIVE FIXATION

Identify the Basic Mechanism involved in Fixation:

Example: Alcoholic fixatives

COAGULANT FIXATIVES

Acts by creating a network that allows solutions to readily penetrate the interior of the tissue.

COAGULANT FIXATIVES

Identify the Basic Mechanism involved in Fixation:

Examples:

1. Zinc salts

2. HgCl

3. Picric acid

4. Ethanol and Methanol

5. Acetone

NON-COAGULANT FIXATIVES

Creates a gel that makes it difficult for fixative to penetrate by subsequent solutions.

NON-COAGULANT FIXATIVES

Must be cut thinly.

1. Speed

2. Penetration

3. Volume

4. Duration of Fixation

PRACTICAL CONSIDERATIONS OF FIXATION:

SPEED

Practical Considerations of Fixation:

• As soon as it is removed from the body

• Done to prevent autolysis and putrefaction

PENETRATION

Practical Considerations of Fixation:

The fixative diffuses into tissue at the rate approximately 1mm per hour, and it slows down as it goes deeper to the tissue

1mm per hour

The fixative diffuses into tissue at the rate approximately ________, and it slows down as it goes deeper to the tissue

PENETRATION

Practical Considerations of Fixation:

• Time of fixation varies with different tissues

• The larger/bigger/harder the tissue, the longer the time that is needed for the fixation

larger/bigger/harder

• The ____ the tissue, the longer the time that is needed for the fixation

VOLUME

Practical Considerations of Fixation:

Amount of fixative is 10-25 times the volume of the tissue to be fixed

10-25 times

Amount of fixative is _____ the volume of the tissue to be fixed

VOLUME

Practical Considerations of Fixation:

Maximum effectiveness of fixation: 20 times the tissue volume (20:1)

20 times the tissue volume (20:1)

Maximum effectiveness of fixation:

DURATION OF FIXATION

Practical Considerations of Fixation:

Fibrous organs need longer fixation than small or loosely textured tissues

Fibrous

_____organs need longer fixation than small or loosely textured tissues

1. Uterus

2. Intestinal tract

Examples of fibrous organs:

DURATION OF FIXATION

Practical Considerations of Fixation:

Fixation time can be cut down by using heat, vacuum, agitation, or microwave

1. Chemical Fixation

2. Vapor Fixation

3. Heat Fixation

4. Microwave Irradiation

5. Ultrasound Fixation

METHODS OF FIXATION:

CHEMICAL FIXATION

Methods of Fixation:

Prevents autolysis by the action of enzymes and deformation of morphologies during specimen preparation

VAPOR FIXATION

Methods of Fixation:

Used to retain soluble materials in situ

HEAT FIXATION

Methods of Fixation:

Involves thermal coagulation of proteins for rapid diagnosis

HEAT FIXATION

Methods of Fixation:

Used for bacteriologic smears

MICROWAVE IRRADIATION

Methods of Fixation:

Use of non-ionizing radiation

MICROWAVE IRRADIATION

Methods of Fixation:

Fixation is due to heat and rapid movement of molecules with the electromagnetic flux

ULTRASOUND FIXATION

Methods of Fixation:

Uses high frequency, high intensity, ultrasonic apparatus

ULTRASOUND FIXATION

Methods of Fixation:

Produces better result and shorter fixation time

• Harden soft and friable tissues

• Make cells resistant to damage and distortion

• Inhibit bacterial decomposition

• Increase optical differentiation of cells and tissue components

• Acts as mordants or accentuators or may inhibit certain dyes

• Reduce risk of infections

GENERAL EFFECTS OF FIXATIVES:

TRUE

TRUE OR FALSE:

Once the tissues are adequately and completely fixed, they are already very safe to handle.

1. Cheap

2. Stable

3. Safe to handle

4. It must kill the cell quickly

5. It must inhibit bacterial decomposition and autolysis

6. It must produce minimal shrinkage of the tissue

7. Permit rapid and even penetration of the tissue

8. It must harden tissues, thereby making the cutting of sections easier

9. It must be isotonic

A good fixative must be:

1. Hydrogen Ion Concentration

2. Temperature

3. Thickness of Section

4. Osmolality

5. Concentration

6. Duration of Fixation

MAIN FACTORS INVOLVED IN FIXATION:

HYDROGEN ION CONCENTRATION

Main Factors Involved in Fixation:

Should always be maintained between at pH 6.0 and 8.0 for optimum fixation of the tissue

pH 6.0 and 8.0

The hydrogen ion concentration should always be maintained between at ____ for optimum fixation of the tissue

40˚C to 45˚C

Many laboratories use tissue processors that work at ____

Room

Surgical specimens: ___ temperature

0-4˚C

Electron microscopy: ideal temperature is between ___

Room temperature

Mast cells: Best react at what temperature even if it is subject to electron microscopy?

HEAT FIXATION

Very common and well known fixation technique in Bacteriology & Blood films

FORMALIN HEATED AT 60˚C

Rapid fixation of every urgent biopsy specimens

FORMALIN HEATED AT 100˚C

For tissues with tuberculosis

small or thin

Tissue blocks taken should be either ____

1 to 2 mm2

Thickness of section in Electron microscopy:

2 cm2, no more than 0.4 cm

Thickness of section in light microscopy:

THICKNESS OF SECTION

This measurement should not be compromised in order to obtain full penetration and satisfactory fixation

10% buffered formalin ; 2-3 weeks ; sectioning

For large solid tissues such as the brain, it is usually suspended whole in _______ for ____ to ensure fixation and hardening prior to ____.

slightly hypertonic solution (400-450 mOsm)

OSMOLALITY:

Best using ______

Sucrose

OSMOLALITY:

Added to Osmium tetroxide fixatives

Hypertonic

OSMOLALITY:

Causes cell shrinkage

Hypotonic

OSMOLALITY:

Causes cell swelling

10% neutral buffered formalin

Most commonly used concentration of formaldehyde:

3% solution

Concentration of glutaraldehyde:

0.25%

Concentration of glutaraldehyde ideal for Immunoelectron microscopy

1 hour

The tissue must be immersed in the fixative no longer than____from the time of interruption of blood flow

2-6 hours

Duration of Fixation:

Using Formalin: should be carried out for ___ during the day the specimen is obtained

3 hours

Duration of Fixation:

Electron microscopy: recommends tissues be fixed for ___, then place in a holding buffer

1. Mercuric chloride and Potassium dichromate

2. Baker’s formol-calcium

3. Imidazole osmium tetroxide

4. Digitonin

LIPID FIXATION:

1. Alcoholic Fixatives

CARBOHYDRATE FIXATION:

Neutral buffered formol saline or formaldehyde vapor

PROTEIN FIXATION:

1. Karnovsky’s paraformaldehyde-glutaraldehyde solution

2. Acrolein

MIXTURE OF FIXATIVES:

Mercuric chloride and Potassium dichromate

Effective for the preservation of lipids in cryostat sections

Baker’s formolcalcium

Preserves phospholipids

Imidazole osmium tetroxide

Post-fixation, improves the ultrastructural demonstration of lipids

Digitonin

Cholesterol fixation for ultrastructural demonstration

Alcoholic Fixatives

• Generally recommended for glycogen fixation

Alcoholic Fixatives

• They can be demonstrated satisfactorily enough for diagnosis

• Although, loss of glycogen can be high which is at 60-80% in aqueous solution

Neutral buffered formol saline or formaldehyde vapor

Are the most commonly used fixative for amino acid histochemistry

Karnovsky’s paraformaldehyde-glutaraldehyde solution

Best known mixture of fixatives

Karnovsky’s paraformaldehyde-glutaraldehyde solution

Best known fixative for electron cytochemistry

Acrolein

Introduced as a mixture of glutaraldehyde or formaldehyde

Acrolein

Penetrates the tissue rapidly

Acrolein

Rapidly preserves the morphology and enzyme activity at low concentration

Acrolein

May also be useful for immersion fixation of surgical biopsies

1. Simple Fixatives

2. Compound Fixatives

TYPES OF FIXATIVE (ACCORDING TO COMPOSITION):