Tissue Processing and Embedding Flashcards
What are the 4 steps of tissue processing
fixation, dehydration, clearing, infiltration
What occurs during dehydration
removal of all unbound water and fixative
What occurs during clearing
removal of the dehydrating agent and provides a solvent for the paraffin wax
What occurs during infiltration
impregnation of the tissue with a support medium
What is the most common dehydrating agent
alcohol
Ethyl alcohol
routinely used, fast acting and non toxic but might cause hardening and shrinkage of tissues
Isopropyl alcohol
miscible with water but not miscible with salt solutions so tissue must be washed following fixation. Can mix with paraffin so clearing agent not always required. Non toxic and does not harden or shrink tissues
Methyl alcohol
similar to ethanol but more hazardous
What is one thing that a clearing agent must do
be soluble in both dehydrants and paraffin
Why must a clearing agent have a high refractive index
to make the tissue appear transparent when mounted on glass
Xylene
routinely used. fast acting and miscible with most solvents and paraffin. Over-exposure will harden tissue. Flamable and moderately toxic
Toluene
similar in action to xylene but unlikely to harden tissues. More volatile than xylene. Most often used in fibromuscular sampels
Chloroform
sometimes used for CNS specimens. Non-flammable but highly toxic, in the presence of oxygen may form phosgene which is exceptionally hazardous
Xylene substitute
less toxic and work almost as well as xylene. Short-chain aliphatic hydrocarbons. Intolerant of water and doesn’t harden tissues
What are universal solvents
capable of dehydrating and clearing tissue
What are the two most used universal solvents
tetrahydrofuran and dioxane
What is infiltration
forms the matrix that prevents distortion of tissue during microtomy
Paraffin wax
the most popular infiltrating and embedding media. non-toxic, inexpensive and easy to use
What are plasticizers used for
to make wax harder and facilitate ribboning
What is beeswax used for
lowers the melting point and makes the sections sticky
What is rubber used for
facilitates ribboning by increasing the elasticity
What are resins used for
makes the wax harder/increases the melting point
What does agitation do during tissue processing
ensures the solutions surrounding the tissue do not become locally saturated and helps avoid dead zones
What does heat do during tissue processing
heat will speed up the process though must be used with caution as using temperatures above 40C can effect the microscopic appearance of the tissue
What does vacuum/pressure do during tissue processing
force solutions into tissues and open porous structures
What does viscosity do during tissue processing
effects how quickly the stations are effective
How does a modern day tissue processor work
tissue remains in a stationary retort and reagent solutions are pumped in and out according to the schedule
What do microwave processors do
speed up processing by heating tissues from the inside which increased the diffusion of solutions. They are formalin and xylene-free
What are the steps for xylene free processing
- fixation
- ethanol dehydration (50% ethanol)
- continued ethanol dehydration (80% ethanol, 20% isopropanol)
- isopropanol clearing (100% isopropanol)
- high temp paraffin wax infiltration
What is a clean cycle
the removal of the paraffin from the retort and lines using xylene followed by alcohol
What is the most common processing issue
incomplete dehydration
What does over processing do to tissues
makes it brittle
What is the most important part of embedding
orientation
How to embed tissues with lumens
so that a cross section can be cut
How to embed tissues with layers
on edge so that all layers are demonstrated
How to embed cassettes with multiple pieces
side by side and oriented so the epithelium is facing the same way
How to embed currettings
clustered centrally but not overlapping
What are acrylic resins used for
to section undecalcified teeth, bones, or to cut thin sections <2um
What are epoxy resins used for
electron microscopy
