2- Fixation and tissue processing

Fixation and unsuitable tissues

•Use of physical and chemical methods to prevent changes associated with tissue decay

•Not suitable for muscle biopsies undergoing enzyme histochemistry

•Skin and mucosal biopsies for investigation of inflammatory skin conditions

What changes occur when a tissue is taken out of the body

•Karyolysis- chromatin breakdown due to DNAases and RNAases

•Nuclear shrinkage and fragmentation

•Autolysis and putrefaction (decay)

Properties of ideal fixative

•Prevent autolysis and bacterial decomposition

•Preserve tissue and its components in their natural state

•Make cellular components insoluble in liquids encountered in tissue processing

•Preserve tissue volume

•Avoid excessive hardness of fixed tissue

•Enhance staining of tissues

•Non-toxic and non-allergenic

Physical fixation methods and their application

•Heating- coagulates proteins e.g. microwave or boiling, used for cell smears and to accelerate chemical fixation

•Freezing- ice crystals form solid matrix e.g. CO2 (dry ice) or liquid nitrogen, used for delicate specimen and frozen sections

Chemical methods of fixation

•Coagulant fixatives- dehydrate tissues, coagulate proteins, rapid penetration but causes shrinkage, poor preservation of mitochondria e.g. ethanol, methanol, acetone

•Form methylene bridges between side chains and end gorups e.g. formaldehyde and glutaraldehyde

•Glutaraldehyde is used for specimens undergoing electron microscopy (good preservation of mitochondria)

Formalin

•10% formalin contains 4% formaldehyde (HCHO)

•Diluted in buffer or saline to form methylene glycol

•Reacts with proteins in sample to form methylene bridges

•Small samples fixed in a few hours, larger overnight

•Exposure monitored, disposal regulated

Fixation rate of formalin

•Penetration rate- time taken to reach innermost part of tissue

•Reaction rate- time taken for tissue to be fully fixed

•Normal rate is approximately 0.75mm per hour

•Cytoplasmic streaming occurs when carbohydrates are pushed to the sides of the tissues, resulting in uneven staining

Other fixatives

•Osmium tetroxide- oxidising agent that acts as a secondary fixative in electron microscopy (black when fixing lipids)

•Mercuric chloride- precipitates proteins, hardens tissues

•Picric acid- alters chargers on protein side chains, good fixative for glycogen, can be explosive

•Potassium dichromate- secondary fixative used for chromaffin reaction in adrenal gland

•Compound fixatives- mixture of fixatives, buffers and salts e.g. B5, zenker’s, carnoy’s

Problems with compound fixatives

•Over and underfixation

•Antigen masking (false negatives)
•Fixation related deposits in tissues

Factors that affect fixation

•Buffers and pH- in a strong acidic environment target groups on proteins become unreactive to formaldehyde and can denature

•Duration and size- must penetrate the entire tissue, compounds in compound fixatives penetrate at different rates

•Temperature- molecules diffuse faster at higher temperatures, tissues enclosed at 60-65 degrees but RT is sufficient

•Concentration of fixative- >10% formalin causes shrinkage and hardening, >70% ethanol causes failure to remove water

•Osmolality of fixative- best if slightly hypertonic, hypotonic shrinks tissues

Decalcification

•Bone samples need calcium removal after fixation to allow for microscopy

•Exception is diagnosis of metabolic bone disease where mineralisation is assesed (embedded in plastic renin instead)

•Acids- disolve calcium salts e.g. nitric acid, HCL

•Chelating agents- bind to calcium ions e.g. EDTA trisodium citrate

What is tissue processing

•Process of removing water from cells and replacing it with a medium that solidifies and allows thin sections to be cut on a microtome

•Following steps are dehydration, clearing (replaces dehydrating agent) and impregnation with supporting medium

Aims of tissue processing

•Embed tissue in a stable, firm, non hazardous medium e.g. paraffin wax

•To facilitate the ability to cut thin sections for microscopy

•To enable good preservation of morphology and cellular contents

•To allow for long term preservation and storage

Dehydration

•Removal of unbound water from the tissues and replacing with alcohol

•Dehydration through ascending grades of alcohol- ethanol, methanol

•Lipids are solubilised- tissue shrinks

•Incomplete penetration will prevent penetration of clearing agents

Factors that affect dehydration

•Mechanical aggitation- increases speed at which alcohol replaces water

•Heat- increases effectiveness of dehydration

•Time- long, slow, dehydration gives the best results, often overnight

•Shrinkage- when dehydration takes place at room temperature and starts with moderate alcohol concentrations, tissue shrinkage is reduced

Clearing for wax infiltration

•Alcohol and wax are not miscible, xylene is fully miscible with both, slides placed in multiple containers of fresh xylene

•Replaces alcohol (dissolves it) in tissue through a process called clearing, impacts optical clarity / transparency of tissue

•Removes a substancial amount of fat from tissue (not suitable when looking for fat)

Properties of clearing agents

•Rapid removal of dehydrating agent

•Minimal tissue damage

•Are flammable, toxic (hydrocarbons) and costly

Examples of clearing agents

•Hydrocarbons- xylene, chloroform, toluene (toxic)

•Terpenes- citrus fruit oils e.g. histoclear (less toxic)

•Xylene substitutes- alkane and limonene based solvents

Infiltration with paraffin wax

•Molten wax displaces xylene, solidifies to consistency tat allows it to be cut by microtome

•Paraffins are a mix of purified wax and additions like plastic resins, antioxidants and dyes

•Heated to 60 degrees, maintains intracellular structures and acts as supporting medium

•Enhanced by vacuum, multiple changes of fresh wax required

•Advantages- not a biohazard, high quality sections, solidifies rapidly, can be used for routine and special stains

Embedding

•Tissue is removed from cassette, placed into mould and surrounded by a molten medium

•Orientation is important e.g. epithelial surface facing up

•Contents need to be checked against biopsy sheet

•Embedding center components- parraffin dispenser, cold plate, heated area for moulds

Typical timings for overnight processing (larger samples)

•10% formalin, 1h, 37 degrees 2x

•50% alcoholic formalin, 1h, 38 degrees

•70% alcohol, 30 mins, 38 degrees

•95% alcohol, 30 minutes, 38 degrees 2x

•100% alcohol, 40 minutes, 38 degrees 2x

•Xylene, 40 minutes, 38 degrees 2x

•Paraffin, 30 minutes, 60 degrees 4x

Rapid processing timeline

•10% formalin for 10 mins at 38ºC. Repeat once.

•70% alcohol for 10 mins at38ºC. Repeat once.

•95% alcohol for 10 mins at 38ºC. Repeat once.

•100% alcohol for 10 mins at 38ºC. Repeat once.

•Xylene for 10 mins at 38ºC. Repeat once.

•Paraffin for 10 mins at 58ºC. Repeat once.