Lecture 3: Tissue processing, de-cal Flashcards
- Define tissue processing
The process of preparing tissue by embedding it in a solid medium for support and rigidity to enable thin sections to be cut.
- What is the main aim of tissue processing
To provide adequate support to the specimen for microtomy
- Give 3 pre-requisites of tissue processing
- Tissue sample must be well fixed
- Tissue sample must be of a suitable size for processing
- Fragile, Friable and Minute fragments must be securede.g.: by wrapping in foam pads.
- Describe a method used for small tissue for ensuring no tissue is lost during processing
Use of a foam pad which goes inside the cassette
- List the 4 major steps in tissue processing in order
1.
- Dehydrating
- Clearing
- Wax Infiltration or Impregnation
- List 4 factors that affect processing
- Type of tissue
- The reagents used
- Agitation
- Heat
- Vacuum
- List the main reagents used in TP
The reagents used..
Dependent on type of investigation.
- How does agitation and heat affect TP
Agitation
- Provides fluid access from all directions.
- Enabling faster and more effective processing
- Define dehydration
Process which displaces the residual fixative as well as cellular water (free not bound).
At the lower end of the ethanol concentrations, water soluble proteins are removed, whilst towards the 100% ethanol step, certain lipids are dissolved.
- What is the purpose of using gradient alcohol in TP
Graded alcohols are used in dehydration to remove free water and keep the bound water in place, as well as to not shock the tissue causing dry and brittle tissue or artifacts
- Give 2 types of dehydrating agents + adv disadv
Ethanol
- hydrophilic i.e.. miscible with water
- however: dissolves lipid; flammable
Iso-propyl alcohol
- xylene free processing
- Irritant, Flammable
Dehydrating agents must meet what four criteria?
Must :
- Replace fixative completely
- Be miscible with fixative reagent
- Not compromise tissue integrity
- Not interfere with subsequent processing step
- Define clearing
Tissue processing step that removes the dehydrant from tissue in preparation for infiltration (De-alcoholization)
- Give advantages and disadvantages of 3 types of clearing agents
Xylene
Most common-cost effective, does not interfere with staining
Causes irritation, toxic, flammable, tissue damage –long term
Chloroform
-slow acting
Causes irritation, toxic-health hazard, causes sectioning difficulty
Toluene
-good tissue structure preservation
Not ideal for routine use-more toxic and more expensive than xylene
- Most common type of infiltrating agent
Paraffin Wax