Expansion
Fixation, dehydration, clearing, embedding, sectioning and staining
Expansion
- Fixation: usually 10 per cent neutral buffered formalin. Cross-links proteins, preventing autolysis and putrefaction. Needs adequate volume and time
- Dehydration: graded alcohols remove water
- Clearing: xylene removes alcohol and makes tissue receptive to wax
- Embedding: molten paraffin wax infiltrates and hardens
- Sectioning: microtome cuts sections of about 4 micrometres
- Staining: dewax, rehydrate, stain, dehydrate, mount
Frozen section bypasses this: tissue is frozen and cut on a cryostat, giving an answer in 15 to 20 minutes during surgery, at the cost of poorer morphology. Used to assess resection margins, confirm malignancy, and identify tissue such as parathyroid.
Fixation artefacts worth recognising: shrinkage, formalin pigment, and poor immunohistochemistry if fixation is prolonged.
Other fixatives: glutaraldehyde for electron microscopy, Bouin solution for testicular tissue, and alcohol for cytology.
Special handling: fresh tissue is needed for lymphoma flow cytometry, cytogenetics, microbiology and for frozen section lipid stains, since processing dissolves lipid.