Tissue Processing & Microtomy Flashcards
Name the 6 sequential steps of histology techniques
Fixation
Dissection/tissue transfer
Processing
Embedding
Sectioning
Staining
Processing steps
After tissue transfer/ sampling/ dissection, cassettes of tissue are ‘processed’
Replace water with wax
Formalin to IMS to Xylene to Wax
Wax provides a suitable supporting medium for sectioning
Embedding steps
When processing complete, cassettes of tissues removed from processor in molten wax
Placed in heated section of embedder
Tissue orientated in a mould of molten wax
Labelled cassette placed on top
Placed on cold place to solidify
Sectioning steps
Thin (2-4µm) sections are cut from the wax blocks on a microtome
Thin enough to permit light to pass through
Sections are ribboned
Tissue sectioning steps
Ribbon of sections floated out on a warm water bath
Minimises creases
Picked up onto glass slides
Ready for staining
Frozen sections
Immediate, intraoperative diagnosis
May be to assess margins or confirm tissue
type
Pathologist or BMS describes the sample and freezes it
Tissue cut on a cryostat– temperature controlled microtome
What are the three main processing steps?
1) Dehydration
2) Clearing
3) Impregnation
Processing - Dehydration
What issues are associated with dehydration?
- Histological tissues are water based
Wax (and resin) are immiscible with water
Alcohol (usually IMS) used as an intermediate reagent
It dehydrates (removes water from) the tissue sections
Usually very effective but not without potential problems:
Can cause shrinkage, make tissue brittle, reduce antigenicity
Too quick and water remains, too slow and tissue is damaged
Processing - Clearing (xylene)
What problems are associated with clearing during processing?
Sections are now dehydrated; but IMS doesn’t mix with wax / (some) resin
A clearing agent is used as an intermediate reagent (or ante-medium)
So-called because it raises the refractive index of the tissue, reducing distortion: Xylene (most commonly used), Chloroform, Toluene, Benzene
Again, usually very effective but not without potential problems:
Too quick and alcohol remains, too slow and tissue is damaged
Processing - Infiltration step ( wax/resin)
Why might tissue need to be infiltrated with resin rather than paraffin wax?
clearing agent is replaced with the embedding medium (wax or resin)
Takes several hours – embedding media are more viscous than the other reagents
Wax needs to be heated so that it is molten, in order to penetrate tissues
Resin generates heat as it forms – heat can damage tissue
Why use wax: harder tissues such as undecalcified bone need a harder supporting medium; to enable thinner sections to be cut (i.e., bone marrow, sections for EM)
What is the purpose of the embedding step?
What tissues may need to be embedded in a particular orientation?
Embedding creates a wax (or resin) ‘block’ in preparation for sectioning
Wax/resin are supportive media which hold the tissues in place for the sectioning step
Some tissues (such as undecalcified bone) need more support than others
Accurate orientation of tissues during embedding is vital
Particular orientation: Skin (epithelia), appendices (longitudinal and transverse views)
Purpose of sectioning
Recommended length/width of sections
Very practical skill, learn by doing
Most FFPE tissues cut at 3 µm
Some tissues need to be cut thinner/thicker than this e.g., Neuro tissues (brain) are cut at 5-6; Lymph nodes are cut at 2µm due to the tissue type and the type of research or diagnostic analysis needed. E.g thinner sections may be needed to visualise individual cells vs overall architecture or gross morphology of a tissue. Also depended on the instrumentation available, as certain equipment will be better use to gain thicker or thinner sections, as well as the sample processing methods as certain methods of fixation and embedding may be better suited to preserve tissue integrity at particular thicknesses. User preference and experience also plays a role.
Can’t reliably get below 2 µm with wax – use resin
What is resin sectioning?
How is it recommended for different tissues?
What are the drawbacks?
Specialist technique
Semi-thin sections for bone marrow
Ultra-thin sections for EM
Very difficult & time consuming
Frozen sections - adv and disadv
Skilled technique
Used intra-operatively or for specialist techniques (IMF)
Advantage: Fast
Disadvantage: Section quality usually poorer than paraffin or resin
