Infiltration / Impregnation and Embedding (F) Flashcards
What is the process (/ steps) in conventional tissue processing (include 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
Tissue processing starts w/ labeling and ends w/ mounting
False, because tissue processing starts w/ labeling and ends w/ labeling
What is the workflow (/ steps) in the histopath lab)?
1) Grossing
2) Freezing
3) Fixation
4) Decalcification (optional)
5) Processing (/ automated tissue processing)
6) Embedding
7) Sectioning
8) Staining (special / routine [H & E staining])
8. 1) LMD
9) Coverslipping
10) Reading
11) Digitalizing
12) Storage
What is infiltration / impregnation?
It is the process of removal of clearing agent
What is the principle (/ action) done in infiltration?
Fill the tissue cavities
Why are the tissue cavities filled in infiltration?
To give a firm consistency
What are the results of infiltration?
1) It promotes easier handling
2) It promotes ease of cutting into thin sections
After infiltration, why is ease of cutting into thin sections achieved?
Due to the firmer consistency present
What are the results if inadequate impregnation is present?
Tissue becomes:
1) Soft
2) Crumbly
Why is infiltration done?
Because tissue blocks crumble when sectioned and break up when floated out in a H2O bath
What are the other terms for embedding?
1) Blocking
2) Casting
What is embedding (/ what is its principle)?
It is the process by w/c the impregnated tissue is placed into a precisely arranged position in a mold containing a medium w/c is then allowed to solidify
What are the 3 concepts associated w/ embedding?
1) Orientation
2) Markers
3) Troubleshooting
What is orientation?
It is how the MT places the tissue (w/c is presented to the patho)
What is the purpose of markers?
These are placed to know the base of the tissues
True or False
The medium used to infiltrate the tissue is usually the same medium utilized for impregnation
True
The medium used to infiltrate the tissue w/c is usually the same medium utilized for impregnation in terms of general purpose is known as what?
Embedding medium / resin
What are the usually used impregnation and embedding media?
1) Paraffin wax
2) Celloidin
3) Gelatin
4) Plastic
What should be the characteristics of an infiltrating and embedding medium?
1) It should be soluble in processing fluids
2) It should be suitable for sectioning and ribboning
3) It is molten between 30 DC and 60 DC
4) It should be translucent or transparent
5) It should be colorless
6) It should be stable
7) It should be homogenous
8) It should be capable of flattening after ribboning
9) It should be non-toxic
10) It should be odorless
11) It should be easy to handle
12) It should be non-expensive
What are the factors affecting infiltration / embedding?
1) Laboratory temperature
2) Number of changes
3) Clearing agent used
4) Volume
What should be the lab temp?
Room temp (20 - 24 DC)
What should be the temp of the paraffin oven?
2 - 5 DC higher than the melting point of the wax
What are the results if the lab temp is beyond 60 DC?
It is deleterious to the tissue; it causes the ff to the tissue:
1) Shrinkage
2) Dryness
3) Brittleness
4) Difficult to section
How many number of changes should be done?
At least 2 changes
How many changes should be done for larger and dense tissues?
More changes
What is the characteristic of chloroform / cedarwood oil (as a clearing agent used)?
It is more difficult to remove
What is the characteristic of xylene / benzene (as a clearing agent used)?
It is easily removed from the tissues
What should be the volume?
25x the embedding medium
True or False
Accdg to Bancroft, low melting point wax typically has higher paraffin concentrations and will provide a softer matrix
True
True or False
Waxes w/ higher polymer content produce a softer matrix w/c will mimic dense tissue more closely
False, because waxes w/ higher polymer content produce a harder matrix w/c will mimic dense tissue more closely
What are the 3 processes of impregnation and embedding process?
1) Manual processing
2) Automatic processing
3) Vacuum embedding and infiltration
What is the manual processing of impregnation and embedding?
1) Impregnation: 4 changes
2) Paraffin wax: 15 mins
3) Paraffin wax: 15 mins
4) Paraffin wax: 15 mins
5) Paraffin wax: 15 mins
6) Embedding (paraffin wax: 3 hrs)
What is the automated processing of impregnation and embedding?
1) Impregnation: 2 - 3 changes w/ agitation
2) 11th (station): melted paraffin wax (1 hr)
3) 12th (station): melted paraffin wax (1 hr)
What is the principle of vacuum embedding and infiltration?
A method of embedding into paraffin under vacuum is proposed. Pcs of tissues are placed into a glass jar containing melted paraffin from w/c the air is pumped out. The jar is permanently placed into TC-80 thermostate
What are the notes (that should be observed) / characteristics of vacuum embedding and infiltration?
1) Negative pressure should be observed (400 - 500 mm Hg)
2) 3 changes should be done
3) It accelerates the processing (if impregnation / embedding process is done)
It is recommended for urgent biopsies, dense & fibrous tissues, connective tissues & delicate tissues
What are the different delicate tissues where vacuum embedding and infiltration is recommended to be used?
1) Lungs
2) Brain
3) Decalcified bones
4) Eyes
5) Spleen
6) CNS
What is the disadvantage of overheating (in vacuum embedding and infiltration)?
It causes:
1) Brittleness
2) Shrinkage
3) Hardening of tissues
What are the different infiltrating / impregnating media?
1) Paraffin wax
2) Paraplast
3) Embeddol
4) Bioloid
5) Tissue mat
6) Ester wax
7) Carbowax
8) Celloidin
9) Low viscosity nitrocellulose
10) Plastic resins
a. Epoxy
b. Polyester
c. Acrylic
11) Gelatin impregnation
12) Agar impregnation
What are the characteristics of paraffin wax?
1) Simplest
2) Most common
3) It is the best embedding medium
4) Has a melting point of 56 - 58 DC
5) When used, incubator should be at 55 - 60 DC / 2 - 5 DC above the melting point (of paraffin wax)
What are the things that should be done when paraffin wax is used as an infiltrating medium?
1) It must be pure and filtered before use
2) It should be only used twice if to be reused
What should be used to filter paraffin wax?
Coarse filter paper
How is H2O removed (if paraffin wax is used)?
H2O is removed by heating the wax to 100 - 105 DC
What are the advantages of using paraffin wax?
1) Serial section may be cut w/ ease w/out distortion
2) It can be used for rapid processing
3) It does not distort the tissue (even if the tissue is immersed for a long time [not 4 hrs >])
4) Staining has good results
What are the disadvantages of using paraffin wax (as an infiltrating medium)?
1) Overheated paraffin makes the sx brittle
2) Prolonged impregnation will cause excessive tissue shrinkage and hardening (hence, it makes the cutting of sections difficult)
3) Inadequate impregnation will promote retention of the clearing agent
4) Tissues that are hard to infiltrate (such as bone, teeth, brains, and eyes) need long immersion for proper support (otherwise, they will crumble on sectioning)
5) Prolonged immersion in paraffin is not advisable
6) Paraffin processing is not recommended for fatty tissues
7) The dehydrating and clearing agents used in the process dissolve and remove fat from the tissues
What are the precautions for paraffin wax impregnation?
1) Prolonged treatment in melted paraffin causes shrinkage and hardening of tissues
2) Infiltration in an overheated paraffin produces shrinkage and hardening
3) Paraffin wax should be pure and filtered
4) For fixed knife microtome, a wax w/ higher melting point should be used
5) In automatic processing, H2O is discarded
6) It is suitable for the ff:
a. Lipid investigation
b. Enzyme (due to the use of heat)
c. Thinner section (for electron microscopy [EM])
d. Undecalcified bone (it is not hard enough for support)
What should be considered for the use of melted paraffin for embedding?
The temp of melted paraffin should be 2 - 5 DC above the melting point of the wax
What is the duration of manual processing of embedding (if paraffin is used)?
3 hrs
What are the ways of cooling for embedding (if paraffin is used)?
1) Refrigerated at -5 DC (if embedding center is absent)
2) Immersion in cold H2O
3) Cold plate in automated processor
What is the composition of paraplast?
Paraffin + synthetic plastic polymer
What is the melting point of paraplast?
56 - 57 DC
What are the advantages of using paraplast (as an infiltrating medium)?
1) It is more elastic and resilient
2) It is good for bones and brain
3) It has same result as double embedding
4) It produces better ribboning sections
5) Serial sections are cut w/ ease w/out cooling the tissue block (no ice crystals artifacts)
6) No deposit is left on the slide after staining
7) No special processing schedule is required
8) It is soluble in common clearing agents
9) It follows the same time schedule for paraffin
What is embeddol?
It is a wax substitute
What are the characteristics of embeddol?
1) It is similar w/ paraplast
2) It is less brittle > paraplast
3) It is less compressible > paraplast
What is the melting point of embeddol?
56 - 58 DC
What is the fxn of bioloid?
It is used for embedding the eyes
What is the characteristic of tissue mat?
It is similar w/ paraplast (but it contains rubber)
What are the characteristics of ester wax?
1) It has a lower melting point
2) It is harder > paraffin
3) It is not soluble in H2O
4) It is only soluble in 95% ethanol and clearing agents
5) If it is used, clearing may be skipped (if clearing is not skipped, it should be done gradually)
For ester wax, if the clearing agent used is cellosolve / xylene, what should be done?
1) Equal parts of clearing agent + ester wax (for 3 - 6 hrs)
2) 3 -4 changes of pure ester wax is done
3) Embedding is done (3 hrs)
4) Section using a heavy duty microtome
What does carbowax contain?
Polyethylene glycol (PEG)
What is the characteristic of PEG?
It is soluble in H2O
What are the characteristics of carbowax?
1) It does not require dehydration and clearing (hence, it can be used for urgent processing)
2) It does not remove neutral fats and lipids
3) If used, the tissue is not exposed to much heat
4) It can be used for enzyme histochemical studies and demonstration of neutral and lipids
What is the disadvantage of using carbowax?
It is hygroscopic
If carbowax is used, what should be observed?
4 changes should be observed, the changes are the ff:
1) 70% carbowax: 30 mins
2) 90% carbowax: 45 mins
3) 2 times at 100% conc at 56 DC: 60 mins
4) For embedding: 50 DC and cooled at ref
What is celloidin?
It is the purified form of nitrocellulose
What is the characteristic of celloidin?
It is soluble in many solvent
What are the uses of celloidin?
1) It can be used for hollow cavities (w/c tend to collapse; ex. eyes)
2) It can be used for cutting into hard tissue (such as bones and teeth)
3) It can be used for whole embryo
4) It is available in various solutions:
a. Thin sections: 2 - 4%
b. Medium sections: 4 - 6%
c. Thick sections: 8 - 12%
What are the advantages of using celloidin?
1) It permits cutting of the tissue section thicker than paraffin wax
2) It can be used for neurological tissues
3) Its rubbery consistency reduces distortion in sectioning
4) If used, no heat is required
What are the disadvantages of using celloidin?
1) It is very slow (takes several wks)
2) If used, thin sections are difficult to cut
3) If used, serial sections are difficult to prepare
4) It is very volatile
5) Blocks and sections must be stored in 70 - 80% alcohol (if not, it will cause discoloration, dryness, and shrinkage)
6) If used, photomicrographs are difficult to prepare
What are the methods of celloidin impregnation?
1) Wet method
2) Dry method
What is the use of wet method?
It can be used for:
1) Bones
2) Teeth
3) Brain
4) Whole organs
What is the process of wet method?
1) Soak in 70 - 80% alcohol (until ready for cutting)
2) Fixation
3) Dehydration
4) Equal parts of ether and alcohol (12 - 24 hrs)
5) Thin sections: 2 - 4% (5 - 7 days)
Medium sections: 4 - 6% (5 - 7 days)
Thick sections: 8 - 12% (3 - 5 days)
6) Embed in a jar / dessicator
7) Store in alcohol
8) Microtomy (/ cutting)
What is the use of dry method?
It can be used for whole eyes
What is the composition of Gilson’s mixture?
Chloroform + cedarwood oil
What is the action of Gilson’s mixture?
It is added to celloidin before allowing it to harden
What is the characteristic of dry method?
There is no need to be stored in alcohol
At what method of celloidin impregnation is Gilson’s mixture used?
Dry method
What is the meaning of LVN?
Low Viscosity Nitrocellulose
What are the characteristics of LVN?
1) It is able to tolerate H2O
2) The sections cut from it are somewhat more easily torn > celloidin sections
3) It is more explosive > celloidin (hence, it should be handled w/ care)
4) When it is dry, it would explode if hit
5) It should be prevented to be exposed to direct sunlight
6) It requires special equipment and conventional microtomy techniques
7) It is not suitable for clinical histology labs
8) Adding tissue plasticizers (such as oleum ricini or castor oil) may prevent tendency of tissues to crack
What are the other names of nitrocellulose (depending on the form)?
1) Cellulose nitrate
2) Flash paper
3) Flash cotton
4) Guncotton
5) Pyroxylin
6) Flash string
What is nitrocellulose?
It is a highly flammable compound
How is nitrocellulose formed?
It is formed by nitrating cellulose through exposure to a mixture of nitric acid and sulfuric acid
What are the uses of nitrocellulose?
It is used in:
1) Rockets
2) Propellants
3) Printing ink bases
4) Leather finishing
5) Celluloid
What is the composition of celluloid?
A mixture of nitrocellulose and camphor
Nitrocellulose + camphor
What is 1st used to manufacture billiard balls?
Nitrocellulose
What are the uses of plastic resins?
1) These are used for light microscope studies
2) These are used for hard tissues
3) These are used for undecalcified bones
4) For high resolution microscopy of tissue sections, these are thinner than usual (4 - 6 um)
5) These are used for renal biopsies
6) These are used for bone marrow biopsies
What are the classifications of plastic resins accdg to their chemical composition?
1) Epoxy
2) Polyester
3) Acrylic
What is the use of epoxy?
It is used in ultrastructural studies (EM)
Why is epoxy used in ultrastructural studies (EM)?
Because the polymerized plastic is sufficiently hard to permit sections as thin as 30 - 40 nm to be cut and it is stable in an electron beam
What are the exs of epoxy?
1) Bisphenol A (/ Araldite)
2) Glycerol (/ Epon)
3) Cyclohexane dioxide (/ Spurr-fasteast infiltration)
What are the things that should be observed if epoxy is used?
1) Reduce antigenicity
2) Sensitization on skin contact and inhalation
3) Toxic components (ex. Viniylcclohexane dioxide)
What is the characteristic of Viniylcclohexane dioxide?
It is carcinogenic
What is the characteristic of bisphenol A?
It is the slowest to infiltrate
What is the characteristic of polyester?
It is rarely used
What is the use of polyester?
It was used for EM but expoxides are more superior
What are the exs of acrylic?
1) Polyglycol methacrylate (GMA)
2) Methyl methacrylate (MMA)
What is the use of GMA?
It is used for light microscopy
Why is GMA used for light microscopy?
Due to the hydrophobic nature
What is the use of MMA?
It is used for:
1) Decalcified bone
2) Other hard tissues
Why is MMA used for decalcified bones and other hard tissues?
Due to hardness
At what circumstances are gelatin impregnation (/ gelatin) used?
1) When dehydration is to be avoided
2) When tissues are to be subjected to histochemical and enzyme studies
What are the uses of gelatin?
1) It can be used for delicate sxs (w/c are friable and necrotic tissues)
2) It can be used for frozen tissue sections
3) It can be used for whole organs (using modification of the Gough-Wentworth technique [accdg to Bankroft])
What are the characteristics of gelatin?
It does not require:
1) Dehydration
2) Clearing
When is gelatin infiltration done?
It is done after washing out of fixative
What is the process of gelatin infiltration?
1) Tissue is placed in 10% gelatin w/ 1% phenol (for 24 hrs)
2) It is then transferred to 20% gelatin w/ 1% phenol (for the next 12 hrs)
3) Finally, it is again transferred to another fresh solution of 20% gelatin w/ 1% phenol
What is the preferred fixative for gel impregnation?
10% formalin (for 12 - 24 hrs)
What is the preferred tissue width for gelatin impregnation?
2 - 3 mm
What is the fxn of 1% phenol (in gelatin infiltration)?
To prevent the growth of mold
What is the preferred volume for impregnation?
25x the tissue
What are the uses of agar impregnation (/ agar)?
1) It is mainly used in double embedding
2) It is also used for fine needle aspiration biopsy (FNAB) sxs (w/c presents small friable pcs)
Why is agar mainly used in double embedding?
Because agar do not prove sufficient support
True or False
If agar is used, multiple fragments and friable tissue may be impregnated in 1 block when sectioning on the cryostat
True
