* Pathology: a study of disease
* Types of tissue specimens
I. Cytological – smear, scrape, brushing, washing or fine needle aspirate
II. Biopsy (Bx) – piece of tissue, trephine, punch specimen
III. Whole organ – amputation or mastectomy, appendectomy
IV. Post mortem samples, routine or forensic
* Tissue preparation modes
i. Routine paraffin blocks
ii. Urgent surgical cases – frozen sections
iii. Enzyme or lipid studies
iv. Immunohitochemistry (antibody)
v. Special tissue studies (kidney, bone, brain)
vi. Electron microscopy
viii. In situ hybridization (DNA or RNA)
* Chemical tissue preservation is used to
i. Prevents tissue breakdown: autolysis, putrefaction
ii. Increases firmness for handling
iii. Retains tissue structure
iv. Increase permeability for future chemical processing
Chemical tissue preservation works:
i. Denatures protein, breaking down autolytic enzymes, unfolding molecules
ii. Disrupting internal bonds (H+ & 2S-) increasing permeability & leaving molecules to make new links with fixatives and dyes
iii. Precipitating proteins and preventing loss during subsequent chemical processing
Important factors of chemical tissue preservation
i. pH (hydrogen ion concentration)
iv. osmolality (critical for electron microscopy)
* Types of tissue preservatives:
i. Aldehydes: Formalin & Gluteraldehyde
ii. Oxidizing agents: osmium tetroxide
iii. Protein denaturing: alcohols
iv. Cross-linking agents: carbodiimides
v. Physical: heat & microwave
vi. Miscellaneous: mercuric choloride
* The volume of fixative should have special consideration, it should be ten times (x10) or greater than that of tissue volume. This ensures no dilution effect by tissue fluids.
* Tissue is processed into paraffin to make it rigid and be able to cut thin slices, so it can be seen through in the light microscope after it has been stained.
* Tissue is processed into paraffin by gradual replacement of water within tissues to finally be in solid paraffin wax
Dehydration in graded alcohols–>alcohol replaced by solvent–>Solvent replaced by wax
Other considerations: agitation and vacuum infiltration
* Standard processing considerations: the use of solvents will remove lipid containing substances, so in stained sections clear areas appeared.
* Paraffin support tissue for cutting, produce a solid block which is easy to store. It can be made by tissue placed into moulds whilst still in molten paraffin –> molten paraffin poured into the mould & left to set
* Lipids cannot be stained since they dissolve in paraffin, so a little hole would appear
* Sectioning paraffin blocks also known as Microtomy.
i. Tissue slices cut between 7-10micrometre on a microtome
ii. Ribbon of sections produced
iii. Ribbons placed on a warm waterbath to partly melt was & smooth out compression wrinkles
iv. Sections separated & mounted on the slides
v. Sections dried for adhesion of tissue
* Deparaffinization of sections are needed because
i. Dyes are water based, so wax needs removal
ii. Unstained sections are opague & tissue structure is obscured
iii. Deparaffinization done through solvent, then decreasing % of ethanol & sections are brought to water
* Staining paraffin sections:
i. Dyes are used to highlight structure
ii. 2 or more contrasting colours are used to help discern different tissue structures
iii. generally dyes either stain nucleic acids or the cytoplasm of cells & surrounding connective tissue
* Routine staining known as H&E (Haematoxylin and eosin). Haematoxylin stains nucleic acids in blue and eosins stains cell membranes and cytoplasm & proteins in pink. Dyes used must contrast with each other, so can discern detail.
* Mounting stained sections are required: to make a permanent record for future reference and legal requirements to be kept for 20 years. This can be done by removing the water from the stained section through increasing % of ethanol & then placed into a solvent. The sections are then drained & a plastic/solvent mixture is dispensed onto a glass coverslip which is then used to cover the tissue, all have the same optical density letting light pass through. This dries produce a permanent record.
* Histotechnology also known as histochemistry, cytochemistry, histological techniques. It defined as the study of intracellular distribution of chemicals, reation sties, enzymes, etc. often by means of staining reactions, radioactive isotope uptake, and selective metal distribution in light & transmission electron microscopy.