Fixation

Fixation

The first and most critical step in histotechnology

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

Primary aim of fixation

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

Secondary aim

• Degeneration

• Decomposition

• Putrefaction

• Distortion

Tissue Cellular process prevented by fixation

Degeneration

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

Decomposition

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

Putrefaction

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

Distortion

Is the alteration of the original shape of a tissue

Autolysis

Fixation prevents ________ by inactivating the lysosomal enzymes. More commonly known as “self digestion”

• Additive Fixation

• Non additive fixation

• Coagulant fixatives

• 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.

Formalin, Mercury, Osmium tetroxide

Example of Additive fixation

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.

Alcoholic Fixatives

Example of non-additive fixation

Coagulant fixatives

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

Zinc salts, HgCl, Picric acid, Ethanol and Methanol, Acetone

Example of Coagulant fixatives

Non-coagulant fixatives

Creates a gel that makes it difficult for fixative to penetrate by subsequent solutions. Must be cut thinly.

• Speed

• Penetration

• Volume

• Duration of Fixation

Practical Considerations of fixation

Speed

Done to prevent autolysis and putrefaction

Penetration

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

Volume

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

20 times the tissue volume (20:1)

Maximum effectiveness of fixation

Duration of Fixation

Fibrous organs need longer fixation than small or loosely textured tissues

Uterus , Intestinal tract

Fibrous organs

• Chemical fixation

• Vapor Fixation

• Heat fixation

• Microwave Irradiation

• Ultrasound Fixation

Methods of fixation

Chemical Fixation

Prevent autolysis by the action of enzyme and deformation of morphologies during specimen preparation

Vapor Fixation

Used to retain soluble materials in situ

Heat Fixation

Involves thermal coagulation of proteins for rapid diagnosis.

Used for bacteriologic smears

Microwave Irradiation

Use of non-ionizing radiation

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

Ultrasound Fixation

Uses high frequency, high intensity, ultrasonic apparatus.

• Hydrogen Ion Concentration

• Temperature

• Thickness of section

• Osmolality

• Concentration

• Duration of Fixation

Main Factors Involved in Fixation

Hydrogen Ion Concentration

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

Room Temperature

Surgical specimen temperature

0-4 degrees C

Electron microscopy ideal temperature

Room Temperature

Mast cells Temperature

Rapid fixation of every urgent biopsy specimens

Formalin heated at 60 degrees C

For tissues with tuberculosis

Formalin heated at 100 degree C

1 to 2 mm²

Electron microscopy tissue thickness

2 cm², no more than 0.4 cm

Light microscopy tissue thickness

Hypertonic

causes cell shrinkage

Hypotonic

Causes cell swelling

10%

Formaldehyde concentration

3% solution

Glutaraldehyde concentration

Shrinkage and hardening of tissue

Prolonged fixation may cause ________ and ______.

• Mercuric chloride and Potassium dichromate

• Baker’s formol-calcium

• Imidazole osmium tetroxide

• Digitonin

Lipid Fixation

Mercuric chloride and Potassium dichromate

Effective for the preservation of lipids in cryostat sections

Baker’s formol-calcium

Preserves phospholipids

Immidazole osmium tetroxide

Post-fixation, improves the ultrastructural demonstration of lipids

Digitonin

Cholesterol fixation for ultrastructural demonstration

Alcoholic fixatives

Carbohydrate Fixation

Alcoholic fixatives

Generally recommended for glycogen fixation

Protein Fixation

Neutral buffered formol saline or formaldehyde vapor is under what kind of fixation?

Neutral buffered formol saline or formaldehyde vapor

Are most commonly used fixative for amino acid histochemistry

Karnovsky’s paraformaldehyde-glutaraldehyde solution

Best known mixture of fixatives and is for electron cytochemistry

Acrolein

introduced as a mixture of glutaraldehyde or formaldehyde. It penetrates the tissue rapidly. May also be useful for immersion fixation of surgical biopsies

• Formaldehyde

• 10% Formol-Saline

• 10% Neutral Buffered Formaldehyde

• Formol-Corrosive

• Alcoholic Formalin

• Alcoholic Formalin

• Glutaraldehyde

Aldehyde fixatives

• simple fixatives

• Compound fixatives

Types of Fixative according to composition

Simple Fixatives

Made up of only one component/substance

Compound Fixative

Made up of two or more fixative

• Microanatomical Fixatives

• Cytological Fixatives

type of fixative according to Action

Microanatomical fixatives

Permit the general microscopic study of tissues structures without altering the structural pattern and normal intracellular relationships of tissues in question

Cytological fixatives

Preserve more specific parts and particular microscope elements of the cell itself

Nuclear Fixative

Are those that preserve the nuclear structures in particular.

Usually contain glacial acetic acid.

Must have a pH of 4.6 or less

Cytoplasmic Fixatives

Preserve cytoplasmic structures

Must NEVER contain glacial acetic acid

Must have a pH of more than 4.6

Histochemical fixatives

Are those that preserve the chemical constituents of cells and tissues

Formaldehyde

Gas produced by oxidation of methyl alcohol.

Pure stock solution (40%)

24 hours

Formaldehyde fixation time

• Reduces basophilic and eosinophilic staining of cells

• Abundant brown pigment granules on blood containing tissues

Formaldehyde if unbuffered

10% Formol-saline

A diluted Formaldehyde with Sodium chloride.

Recommended for Central Nervous tissues.

10% Neutral Buffered Formaldehyde

Also known as Phosphate-Buffered Formalin

Had a pH maintained at 7.0

Sodium dihydrogen phosphate, disodium hydrogen phosphate , 40% Formaldehyde, and distilled water

Composition of 10% NBF

Formol-Corrosive

Also known as “Formol-sublimate”

Penetrates small pieces if tissues rapidly.

No need for “washing out”

Alcoholic Formalin

also known as “Gendre’s fixative”

• Used for faster diagnosis because it fixes and dehydrate at the same time

Causes partial lysis of RBC

Disadvantage of alcoholic formalin

Glutaraldehyde

Made up of two formaldehyde residues, linked by three carbon chains

• Mercuric Chloride

• Chromate fixatives

• Lead fixatives

Metallic Fixatives

Mercuric chloride

Most common metallic fixative, used in saturated aqueous solutions of 5-7 %, widely used as a secondary fixative

Zenker’s fluid

Made up of mercuric chloride stock solution for which glacial acetic acid has been added.

Recommended for fixing small pieces of liver, spleen, connective tissue fiver and nuclei.

Recommended for trichrome staining

Dezenkerization

Mercury deposits may be removed by ?

Zenker-formol

Also known as “Helly’s solution”

Excellent Microanatomic fixative for pituitary gland, bone marrow, and blood containing organs such as spleen and liver

Heidenhain’s Susa solution

Recommended mainly for tumor biopsies especially of the skin and an excellent cytologic fixative

B-5 fixative

Commonly used for bone marrow biopsies

Chromic acid, Potassium dichromate, Regard’s fluid, Orth’s fluid

Chromate fixatives

Chromic acid

• Used in ½ % aqueous solution

• A constituent of a compound fixative

• Precipitates all proteins and adequately preserves carbohydrates

• A strong oxidizing agent

Potassium dichromate

• Used in 3% aqueous solution

• Preserves lipids

• Preserves mitochondria (pH 4.5 - 5.2)

Regard’s fluid

Also known as “Muller’s fluid”

• recommended for the demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid-containing tissues.

• Does not preserve fat

Orth’s fluid

Recommended for study of early degenerative processes and tissues necrosis.

Demonstrates rickettsia and other bacteria

Lead fixatives

Used in 4% aqueous solution of basic lead acetate.

Recommended for acid mucopolysaccharides

• Bouin’s solution

• Brasil’s alcoholic picroformol fixative

Picric acid fixatives

Picric acid

Preserves glycogen but can cause shrinkage of tissue. Suitable for analine stains.

May give yellow color to the tissue

Bouin’s solution

Recommended for fixing embryo’s and pituitary biopsies.

Brasil’s alcoholic picroformol fixative

Better and less messy than Bouin’s solution.

It is an excellent fixative for glycogen

Glacial acetic acid

• Fixes and precipitates nucleoproteins

• Precipitates chromosomes and chromatin materials

• Solidifies at 17 degrees C

• Methyl alcohol

• Isopropyl alcohol

• Ethyl alcohol

• Carnoy’s fluid

• Newcomer’s fluid

Alcohol fixatives

Alcohol fixatives

Denatures and precipitates proteins

Used both as a fixative and dehydrating agent

Methyl Alcohol

Excellent for fixing dry and wet smears, blood smears, and bone marrow tissues

Isopropyl alcohol

(pero ingon si miss aniñon methyl alcohol kuno answer😒)

Used for fixing touch preparations

methyl alcohol

Fixes blood, tissue films and smears

• preserves nucleoproteins, nucleic acids

Carnoy’s fluid

Recommended for fixing chromosomes, lymph glands, and urgent biopsies.

Most rapid fixative

Also used to fix brain tissue for diagnosis of rabies

Newcomer’s fluid

Recommended for fixing mucopolysaccharides and nuclear proteins