Tissue Processing

Question 1

What is the aim of tissue processing

Answer 1

To embed the tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut, and yet soft enough not to damage the knife
The most satisfactory embedding material for routine histology is paraffin wax
Most fixatives are aqueous based and these are not miscible with paraffin wax, to enable impregnation with wax the tissu must therefore by processed

Question 2

What are the four stage of tissue processing

Answer 2

Dehydration
Clearing
Impregnation
Embedding

Question 3

What is the dehydration stage of processing?

Answer 3

To remove fixative and water from the tissue and replace the with dehydrating fluid
There are numerous dehydrating agents and they are generally used as increasing strengths
Using IMS tissue I immersed first in 70% IMS in water and then processed through 90% then finally to 100%
In this way all the aqueous tissue fluids are removed but with little disruption to the tissue
Examples of some dehydrating agents include ethanol, IMS, methanol and acetone

Question 4

What is the clearing stage of processing

Answer 4

Replacing the dehydrating agent with a fluid that is totally miscible with both the dehydrating agent and the embedding medium (wax)
Few dehydrating agents are mscible with paraffin-> they need to be hydrocarbon solvents
Examples = xylene, toluene, histoclear etc

Question 5

What is the impregnation stage of processing

Answer 5

Replacing the clearing agent with the embedding medium.
Paraffin wax is the most popular embedding medium for histology

Question 6

What is the emedding stage of processing

Answer 6

Placing the tissue in a wax block to facilitate further testing

Question 7

What factors influence the rate of tissue processing

Answer 7

Agitation
Heat
Viscosity
Vacuum

Question 8

How does agitation affect tissue processing

Answer 8

Most modern processors involve agitation to allow for uniform and faster processing of samples

Ensures maximum surface area exposure

Question 9

How does heat affect tissue processing

Answer 9

Introducing a higher temp speeds up the penetration of the chemicals

Question 10

How does viscosity affect tissue processing

Answer 10

Viscosity is the internal friction of a particular substance which affects rate of flow through tissues and is inversely proportional to temp
It is particularly important in the clearing and infiltration stages of tissue processing
Substances with high molecular weight, such as some transition solvents and waxes have high viscosity and diffusethrough tissues more slowly than thos of lower molecular weight

Question 11

How does vaccuum affect tissue processing

Answer 11

Vacuum improves the quality of processing

Question 12

What are the benefits to automated tissue processing

Answer 12

Reduced TATs by > 24 hrs
Constant agitation means superior results

Question 13

Why might you see a fresh specimen in the lab

Answer 13

For frozen sectioning
For lipid special stains e.g. oil red o
Sample meant for immunology for Igs
No formalin available in theatre
For glutaraldehyde/for sending out to Beaumont for electron microscopy