Fixation and Fixatives

FIXATION AND FIXATIVES

AIMS OF FIXATION Prevention of putrefaction and autolysis

of tissues Prevention of change of shape or

volume during any subsequent procedures

Maintainance of tissues in a state close to their normal living state

Solidification of colloid material of cells and hardening of tissues

DISADVANTAGES OF FIXATION Molecular loss Swelling/shrinkage Variation in staining Accuracy of biochemical analysis

Capacity to maintain intracellular organelles

FACTORS EFFECTING FIXATION

pH Temperature Thickness of the tissue section

Osmolality of solution Duration of fixation additives

TYPES OF FIXATION PHYSICAL-HEAT - MICROWAVE - FREEZE DRYING

CHEMICAL

CLASSIFICATION

Simple Compound

Microanatomical fixativeCytological fixativeHistochemical fixative

REAGENTS EMPLOYED AS FIXATIVES Formaldehyde

40% formaldehyde= formalin cross links between proteins-?? fixes phosholipids and unsaturated fatty acids

Advantages- easily available cheap penetrates and fixes tissues well preserves fat, myelin, nerve fibres, amyloid, haemosiderin, organisms

FORMALIN Disadvantages paraformaldehyde slow dermatitis, irritating to eyes impurity of formic acid iron and other pigments changes colour of bile precipitates fine crystalline pigment

from hemoglobin REVERSIBILITY

GLUTARALDEHYDE MOA- cross link-?? Advantages- - ultrastructure Thickness of section IHC staining

MERCURIC CHLORIDE MOA- ammonium salts, amides, amines Rapid penetration and hardening Cytoplasmic and nuclear staining Poor depth of penetration Shrinkage Overhardening Radio-opaque Corrodes metals

POTASSIUM DICHROMATE Good cytoplasmic stain Preserves phospholipids Moderate hardening Mordant for staining Poor nuclear stain Wash in water overnight before transfer

to alcohol

CHROMIC ACID Powerful oxidising agent Preserves carbohydrates Orth’s fluid and zenker’s fluid

OSMIUM TETRAOXIDE 2% stock solution Lipids like myelin –black-?? Cytological fixation Expensive Poor and uneven penetration Irritating to eyes ultrastructure

PICRIC ACID Explosive when dry Precipitates proteins as picrates Trichrome stain gives a good contrast

ETHYL ALCOHOL Fixing blood smears Carnoy’s fluid Precipitates protein and glycogen is

preserved Demonstration of enzymes Dissolves fat and lipids

ACETONE Histochemical demonstration of

enzymes phosphatases and lipases

ACETIC ACID Swelling of tissues Good nuclear fixation Poor cytoplasmic fixation

TRICHLOROACETIC ACID Swells tissues Protein precipitant Decalcifying properties

MICROANATOMICAL FIXATIVESFORMAL SALINE 100 ml formalin 900 ml tap water 8.1 g NaCl Tolerant fixative 3 mm thick-56°C-3 hrs Neutral buffered formalin?? Formal zinc??

FORMAL CALCIUM 90 ml water 10 ml formalin 2 g calcium chloride preserves phosholipids Not recommended if calcium, acid or

alkaline phosphatases have to be estimated.

HEIDENHAIN SUSA HgCl2 NaCl Formalin Trichloroacetic acid Glacial acetic acid Distilled water Best fixative for routine biopsy Intolerant fixative Good cytological staining

ZENKER’S FLUID HgCl2 Potassium dichromate Sodium sulfate Distilled water Glacial acetic acid Bloody specimen Trichrome stain Mallory’s connective tissue stain

HELLY’S FLUID Zenker’s+formalin For hematologic tissues If giemsa/ leishman stains

BOUIN’S & GENDRE’S FLUID Picric acid Formalin Glacial acetic acid Connective tissue stain Glycogen preservation-RCC, liver, muscle

CYTOLOGICAL STAINSCARNOY’S FLUID

ABSOLUTE ALCOHOL CHLOROFORM GLACIAL ACETIC ACID RAPID NUCLEAR FIXATION GLYCOGEN PRESERVATION

NEWCOMER’S FLUID ISOPROPANOL PROPIONIC ACID PETROLEUM ETHER ACETONE DOXANE RAPID FIXING CHROMOSOME

CHAMPY’S FLUID POTASSIUM DICHROMATE CHROMIC ACIID OSMIUM TETRAOXIDE Slow uneven penetration Cytological fixation Preserves fat, lipids