Clearing (F) Flashcards
What is the process (/ steps) of conventional tissue processing (including all minor steps)?
1) Labeling (Numbering)
2) Fixation
2. 1) Washing out
3) Decalcification (optional)
4) Dehydration
5) Clearing
6) Impregnation (Infiltration)
6. 1) Orientation
7) Embedding
7. 1) Trimming
8) Section-Cutting (Microtomy)
8. 1) Floating Out
8. 2) Adhesion
9) Staining
10) Mounting
10. 1) Ringing
11) Labeling
True or False
Conventional tissue processing starts w/ labeling and end w/ labeling
True
What is the other term for clearing?
Dealcoholization
What is the rationale of clearing / dealcoholization?
Although the dehydrated tissue is now essentially H2O-free, it still cannot be infiltrated w/ wax because wax and ethanol are largely immiscible
What is needed to remove alcohol and other dehydrating solutions from tissues prior to embedding and from finished slides prior to mounting?
An intermediate solvent that is fully miscible w/ both ethanol and paraffin wax
What is the usual intermediate solvent that is needed to remove alcohol and other dehydrating solutions from tissues prior to embedding?
Paraffin wax
What is clearing?
It is the process of removal of dehydrant
What must be the characteristics of a clearing agent?
1) It must be fully miscible to ethanol
2) It must be fully miscible to paraffin wax
What are the actions of clearing?
1) It imparts an optical clarity or transparency to the tissue due to their relatively high refractive index (RI)
2) It removes a substantial amt of fat from the tissue w/c otherwise presents a barrier to wax infiltration
When is clearing done?
1) After dehydration
2) Or before infiltration
3) After staining
4) Or before staining
What should be the volume of the clearing agent for tissues that are 4 mm thick?
20x
How many lvls of xylene are done (and their corresponding durations) in the clearing sequence of tissues that are 4 mm thick?
2 lvls
Xylene - 1 hr
Xylene - 1 hr
How many lvls of xylene (accdg to Gregorios; and their corresponding durations) are used for clearing sequence of 4 mm thick sxs?
2 lvls
Xylene - 25 mins
Xylene - 45 mins
When is complete clearing observed?
When the internal structures become visible to the naked eye
Prolonged clearing will cause what?
1) Brittleness
2) Tissue becomes more difficult to cut
What is the name of the processor (/ machine) used for automated tissue processing?
Leica TP 1020
Leica TP 1020 is used for what time duration?
Overnight (12 hrs)
What are the sxs that can be processed via the use of Leica TP 1020?
General surgical sxs
What are the characteristics of the sxs that can be processed via the use of Leica TP 1020?
1) 3 mm thick
2) < or equal to 20 x 20 mm
How many stations are present in Leica TP 1020?
12 stations
What are the 12 stations (what are the rgnts used and the corresponding time for each station) present in Leica TP 1020?
1) NBF: 1 hr + hold time
2) 95% alcohol: 1 hr
3) 95% alcohol: 1 hr
4) 95% alcohol: 1 hr
5) 100% alcohol: 1 hr
6) 100% alcohol: 1 hr
7) 100% alcohol: 1 hr
8) Xylene: 1 hr
9) Xylene: 1 hr
10) Xylene: 1 hr
11) Melted paraffin: 1 hr
12) Melted paraffin: 1 hr
What is a pro-par clearant?
It is a unique aliphatic substitute for xylene
What are the characteristics of pro-par clearant?
1) It is non-irritating
2) It is non-sensitizing
3) It is greaseless
4) It is practically odorless
5) It is not derived from citrus oil
6) It is recycable
What are the characteristics of a good clearing agent?
1) It should be miscible w/ alcohol
2) It should be miscible w/ paraffin
3) It should be miscible w/ mounting media
4) It should make the tissue transparent
5) It should not produce shrinkage
6) It should not produce hardening
7) It should not produce damage
8) It should not dissolve aniline dyes
9) It should not quickly evaporate in H2O bath
Provide an ex. of aniline dyes
Eosin
What are the factors affecting clearing?
1) Boiling point
2) Viscosity
3) Refractive index
True or False
Agents w/ low boiling point are readily replaced by paraffin
True
In accordance to the principle of boiling point (as 1 of the factors that affects clearing), what is the only agent that is not readily replaced by paraffin?
Chloroform
What is the principle of viscosity (as a factor that affects clearing)?
Higher viscosity leads to slow penetration
What does refractive index (as a factor that affects clearing) affect?
It affects the quality of cleared tissue and it does not affect the rate (of clearing)
What are the different clearing agents?
1) Xylene / Xylol
2) Toluene
3) Benzene
4) Chloroform
5) Cedarwood oil
6) Aniline oil
7) Clove oil
8) CCL4
9) Dioxane
10) Tetrahydrofuran
11) Terpenes
12) Methyl benzoate / Salicylate
13) Carbo-xylene
What are the advantages of using xylene (as a clearing agent)?
1) It is the routine clearing agent
2) It is most rapid
3) It is a common clearing agent
4) It is suitable for urgent biopsies (since it is a most rapid clearing agent)
5) It could be used for celloidin sections
6) It evaporates quickly in paraffin oven (hence, it can be readily replaced by wax during impregnation and embedding)
7) It is cheap
Since xylol is the most rapid clearing agent, what is the duration of its clearing when it is used for urgent biopsies?
Within 15 - 30 mins
What are the other uses of xylene?
1) It can be used as a dewaxing agent (during staining)
2) It can be used in cover slipping
3) It can be used in cleaning tissue processors
4) It can be used as a solvent (to remove synthetic immersion oil from the microscope objective)
5) It can be used for recycling used slides
What are the advantages of using toluene (as a clearing agent)?
1) It is better at preserving the tissue structure
2) It is tolerant to small amt of H2O
3) It can be used as a substitute for xylene or benzene
4) It is miscible in absolute alcohol
5) It is miscible in paraffin
6) If the tissue is left in toluene (for 24 hrs), the tissue does not become excessively hard and brittle
7) It has 1 - 2 hrs clearing time
8) It clears overnight
9) It is not carcinogenic
What are the disadvantages of using toluene (as a clearing agent)?
1) It is slower > xylene and benzene
2) It tends to acidify the pH
3) If highly concentrated solutions of toluene is used, it will emit fumes
4) It is more expensive
What is the characteristic of the fumes emitted if highly concentrated solutions of toluene is used?
The fumes emitted are toxic (upon prolonged exposure)
What are the advantages of using benzene (as a clearing agent)?
1) It is rapid acting (hence, it is recommended for urgent biopsies: because it has a clearing time of 15 - 60mins)
2) It volatilizes rapidly in paraffin oven (hence, it is easily eliminated from the tissue)
3) It is miscible w/ absolute alcohol
4) It does not make tissues hard and brittle
5) It causes min. shrinkage
6) It makes tissues transparent
What are the disadvantages of using benzene (as a clearing agent)?
1) It is highly flammable
2) If a tissue section is left in benzene (for a long time), considerable tissue shrinkage may be observed
3) It is extremely carcinogenic to man (because it causes aplastic anemia)
What are the advantages of using chloroform (as a clearing agent)?
1) It causes less brittleness > xylene
2) It is recommended to be used for tough tissues, nervous tissues, lymph nodes, and embryos
3) It is suitable for large tissue sx
4) It is not flammable
5) It is miscible w/ absolute alcohol
What are the exs of tough tissues where chloroform is recommended to be used?
1) Skin
2) Fibroid
3) Decalcified tissues
What are the disadvantages of using chloroform (as a clearing agent)?
1) It is toxic to the liver (hence, it should be used in a well ventilated room)
2) The infiltration of wax is low
3) It becomes milky in prolonged storage (hence, it must be filtered)
4) Its vapor may attack the rubber seal used in vacuum impregnating bath
5) It is difficult to be removed from paraffin sections (it may even produce considerable deterioration of the wax)
6) Complete clearing is difficult to evaluate
7) The tissue tends to float
6)
What are the advantages of using cedarwood oil (as a clearing agent)?
1) It clears both paraffin and celloidin section
2) It is recommended to be used for CNS tissues and cytological studies (particularly of smooth muscles and skin)
3) 2 changes of solution are used
4) It often improves cutting of the sections
5) Tissues may be left in cedarwood oil indefinitely
6) It does not dissolve out aniline dyes
7) It makes the tissue transparent
8) It does not harden tissues
9) It does not interfere too seriously w/ paraffin penetration (if it is not completely removed)
What are the disadvantages of using cedarwood oil (as a clearing agent)?
1) Its clearing time is extremely slow
a. 2 - 3 days (for paraffin)
b. 5 - 6 days (for celloidin)
2) In terms of penetration, it is slightly slower > benzene
3) It is hard to eliminate from the tissues in paraffin bath
4) The tissues cleared in cedarwood oil initially float before gradually staying to the bottom as clearing proceeds (hence, the tissue may out before it is completely cleared)
What should be done to improve or hasten the elimination of cedarwood oil from the tissues (w/c are in paraffin bath)?
Transfer the sx from cedarwood oil to benzene for 1/2 hr before finally placing the tissue in wax
When the tissues are cleared in cedarwood oil, these initially float before gradually staying to the bottom as clearing proceeds (hence, the tissue may out before it is completely cleared), how can this circumstance be prevented?
1) This can be prevented by superimposing absolute alcohol on the surface of the clearing agent
2) Once saturated, the sx should then be transferred to a fresh solution of cedarwood oil
What are the disadvantages of using cedarwood oil (as a clearing agent)?
1) It becomes milky (upon prolonged exposure), hence, it should be filtered before use
2) It is expensive
3) Cedarwood oil that has been previously used to clear acetic-alcohol fixed tissues may produce crystals w/ a melting point of approx 35 DC, hence, it will interfere w/ the adequate clearing of tissue
Since cedarwood oil that has been previously used to clear acetic-alcohol fixed tissues may produce crystals w/ a melting point of approx 35 DC, hence, it will interfere w/ the adequate clearing of tissue, what should be done to this circumstance?
The solution must be heated to 200 DC to dissolve the crystals
What is the fxn of aniline oil (as a clearing agent)
It is recommended to be used for:
1) Embryos
2) Insects
3) Delicate sxs
What is the action of clove oil (as a clearing agent)?
1) It makes tissue brittle
2) If used, aniline dyes are removed
3) If used, celloidin is dissolved
What is the other term for CCL4?
Carbon tetrachloride
What are the characteristics of CCL4 (as a clearing agent)?
1) Its characteristics are same w/ chloroform
2) However, it is cheaper > chloroform
3) It is highly toxic
What are the 2 uses of dioxane?
1) Graupner’s
2) Paraffin
How is dioxane used for Graupner’s?
3x in 4 hrs (/ 3 changes of dioxane for 4 hrs)
How is dioxane used for paraffin?
3x in 90 mins
What are the characteristics of tetrahydrofuran (as a clearing agent)?
1) It is superior to dehydrating and clearing agents
2) It is non-toxic
3) It has an offensive odor
What are the exs of terpenes (as a clearing agent)?
Citrus oils such as:
1) Essential oils
2) Terpineol
3) Cedarwood
4) Clove
5) Lemonene
6) Oreganum
7) Sandal wood
Where did terpineol came from?
It came from liliac oils of bergamot
What is the fxn of methyl benzoate / salicylate (as a clearing agent)?
It is recommended to be used for double embedding
What is the fxn of carbo-xylene?
It is used for thick mucinous Papanicolaou smears
