MODULE 2 - Decalcification & Dehydration/Clearing Flashcards
Removal of calcium ions or lime salts from the organic extracellular matrix, calcified collagen and surrounding tissues of bones A. Decalcification B. Fixation C. Dehydration/Clearing D. Embedding
A
Both decalcification and processing depend on
Bone thickness
Ideal thickness of bone tissues
1-3 mm
Decalcification must be done following
Fixation
Ratio of fluid to tissue in decalcification
20:1 or 10-20x the volume of tissue
Factors that can speed up decalcification
Heat and agitation
This can decalcify more rapidly but may destroy tissue.
Concentrated acid solutions
Optimal temperature in decalcification
Room temperature
Decalcification can be done on autotechnicon
True or false
False, it must be done MANUALLY
It is not usually done and not suited on urgent biopsies
A. Electrolytic method and Ion exchange resin
B. Use of acid and use of chelating agent
C. Ion exchange resin and use of chelating agent
C
It Inactivates alkaline phosphatase activity
Use of chelating agent: EDTA
A combination of nitric acid and formaldehyde
Formol nitric acid
It requires fumehood.
Formol nitric acid
Duration of ion exchange resin
1-14 days
Use of chelating agent: EDTA is best for
EM and immunohistochemistry
Calcium is rapidly removed by the decalcifying solution containing formic acid, thereby increasingly solubility from tissues A. Ion exchange resin B. Electrolytic method C. Use of Acid D. Use of chelating agent: EDTA
A
The positively charged calcium ions are attracted to negative electrodes from the decalcifying solution A. Ion exchange resin B. Electrolytic method C. Use of Acid D. Use of chelating agent: EDTA
B
Duration for small specimen in using chelating agent: EDTA
1-3 weeks
Duration for dense tissue in using chelating agent: EDTA
6-8 weeks
Duration in using Ion Exchange resin.
1-14 days
Use of Chelating agent: EDTA inactivates alkaline phosphatase activity, what is the remedy?
Add magnesium chloride
In ion exchange resin, tissue must be placed at the bottom of container with what to increase solubility from tissues
ammonium form of polystyrene resin
The most rapid method of decalcification A. Ion exchange resin B. Electrolytic method C. Use of Acid D. Use of chelating agent: EDTA
B
It is not routinely used because it is weak and slow. A. Nitric acid and formol nitric acid B. Von ebner's C. HCl and chromic acid w/ flemming's D. HCl and TCA and sulfurous acid
D
4 solutions under Nitric acid
10% aqueous nitric acid
Formol nitric acid
Perenyi, chromic acid and ethyl alcohol
Phloroglucin nitric
Most rapid type of nitric acid A. 10% aqueous nitric acid B. Formol nitric acid C. Perenyi, chromic acid and ethyl alcohol D. Phloroglucin nitric
D
It is commonly used but should not be used in concentrated solutions
Nitric acid
It contains chloroform as preservative
Citric acid citrate buffer
It is highly corrosive on skin and is considered as an environmental toxin
chromic acid and flemming’s w/ HAc
Used for cartilage, research, autopsy and BM specimen
Formic acid sodium citrate
Duration when using 10% aqueous nitric acid
24 hrs
Duration of using Formol nitric acid
1-3 days
Duration of using Perenyi’s nitric acid, chromic acid and ethnyl alcohol
2-7 days
Perenyi’s nitric acid, chromic acid and ethnyl alcohol all are:
2-in-1 decalcifying and softener
HCl is used for
Von Ebner’s is used for:
minute pcs of bones
teeth and small pcs of bones, surface decalcification of blocks
Physical / mechanical method is done by
bending, probing and pricking tissue with needle
Physical/mechanical method is reliable
True or false
False
The easiest METHOD OF TESTING FOR THE COMPLETENESS OF DECALCIFICATION A. Physical/Mechanical B. X-ray/Radiological C. Bubble Test D. Chemical/Calcium oxalate
A
METHOD OF TESTING FOR THE COMPLETENESS OF DECALCIFICATION
It is done by adding calcium carbonate
Bubble test
Can detect even the smallest amount of calcium
X-ray/radiologic
Disadvantage of using X-ray / Radiologic Method
cannot be used on mercuric chloride fixed tissues
The most reliable and accurate method A. Physical/Mechanical B. X-ray/Radiological C. Bubble Test D. Chemical/Calcium oxalate
B
In using x-ray method, appearance of this will indicate incomplete decalcification
opaqueness in x-ray films
In using chemical/calcium oxalate test, what will you add to make it alkaline?
Strong ammonia
You can check the solution if its alkaline by using
litmus paper
In using chemical/calcium oxalate test, what will be added after it becomes alkaline?
ammonium oxalate
In using chemical/calcium oxalate test, this result will indicate incomplete decalcification
Cloudiness
In using chemical/calcium oxalate test, for how long will you allow the solution to stand?
30 mins
In using chemical/calcium oxalate test, how many mL of discarded fluid will you put in the tube?
5 mL
Decalcified tissues are neutralized by
(1) Immersing in saturated lithium carbonate or 5-10% sodium bicarbonate
(2) Rinsing in tap water
(3) Storing in formol saline with 15% sucrose or PBS with 15-20% sucrose at 4 degC
Enumerate the tissue softeners used for unduly hard tissues
(1) 2% HCl or 1% HCl in 70% alcohol
(2) Perenyi’s fluid
(3) Molliflex fluid
What is lendrum’s method?
It is used for hard tissues like cervix and it is done by immersing tissues in 4% aqueous phenol for 1-3 days
Decalcification is mandatory
True or False
False
Dehydration is mandatory
True or False
True
In dehydration, we may use autotechnicon
True or False
True
It pertains to the removal of intercellular and extracellular water from tissues after _____ and prior to_____.
Fixation and wax impregnation
In dehydration, concentration used in delicate tissues like embryo may be
30% concentration
Dehydration uses _______ of alcohol.
Ascending/increasing concentration
In dehydration, the amount of dehydrating agent must be
not less than 10x the volume of specimen
In dehydration, the initial concentration for routine is
between 70-80%
In dehydration, low concentration tends to
macerate the tissues
In dehydration, higher/95% concentration will
harden only the surface, deeper parts will not be penetrated
This is used as routine dehydrating agent, FAST and NON-TOXIC A. Ethanol B. Methyl alcohol C. Butyl alcohol D. Pentanol
A
It can be used as ethanol and xylene substitute, usually used for microwave techniques A. Ethanol B. Methyl alcohol C. Butyl alcohol D. Isopropanol/IPA
D
It is not commonly used because it is toxic. A. Ethanol B. Methyl alcohol C. Butyl alcohol D. Pentanol
B
Methyl alcohol as dehydrating agent can only be used for
blood and tissue films
It is used for plant and animal microtechniques A. Ethanol B. Methyl alcohol C. Butyl alcohol D. Pentanol
C
It dissolve paraffin. A. Ethanol B. Methyl alcohol C. Butyl alcohol D. Pentanol
D
Enumerate dehydrating agents under alcohol
- Ethanol
- Methyl alcohol
- Butyl alcohol/butanol
- Isopropanol/IPA/isopropyl alcohol
- Tertiary butanol
- Pentanol
In dehydration, prolonged storage in alcohol will
affect staining
Dehydration can be hasten when you apply ___ @____
heat @37 degrees
It can be used too accelerate dehydration process and acts as an indicator for water saturation
Anhydrous copper sulfate
Where will you put anhydrous copper sulfate to accelerate dehydration process?
at bottom of the container
In using anhydrous copper sulfate, this will indicate full saturation of dehydrating fluids with water.
Bluish discoloration
Disadvantage of using acetone
evaporates easily and highly flammable
It is fast-acting and is used for urgent biopsies, but NOT ROUTINELY USED.
Acetone
Cellosolve is also known as
ETHYLENE GLYCOL MONOETHYL ETHER
Main disadvantage of using Diethyl dioxide/dioxane
extremely dangerous and toxic to man
Dioxane is also known as _____ and its 2nd disadvantage is
Diethylene dioxide, it ribbons poorly
Dioxane can act both as ___ and ____
dehydrating and clearing agent
2 Methods of Dioxane dehydration
Graupner’s and Weisenberger’s
Cellosolve is combustible @ what temperature
110-120 degrees fahrenheit
In Weisenberger’s – tissue wrap in gauze bag and placed in a bottle with _______ and ______________.
Dioxane and calcium oxide/quicklime
Cellosolve decomposes upon exposure to _______.
sunlight
ETHYLENE GLYCOL MONOETHYL ETHER is toxic to:
inhalation, ingestion, skin contact (prolonged exposure)
fetal, urinary, reproductive and blood systems
It is used for sections and smears and it causes minimum shrinkage
Triethyl phosphate
Triethyl phosphate causes _________
minimum shrinkage
Both dehydrating ang clearing agents
A. Diethylene dioxide and Tetrahydrofuran
B. Ethylene glycol monoethyl ether and Triethyl phosphate
C. Diethylene dioxide and Triethyl phosphate
A
It has offensive odor and may cause conjunctival irritation
Tetrahydrofuran (THF)
Tetrahydrofuran is _______ and can cause
nausea, dizziness, headache and conjunctival irritation
A fast acting and a routine clearing agent
xylene
Can be a substitute for xylene and benzene
Toluene
Xylene becomes _______ when an incompletely dehydrated tissue is immersed in it
milky
rapid clearing time of xylene is
30 mins - hrs
clearing time of toluene
1-2 hrs
Xylene is not suitable for
lymph nodes and nervous tissue
disadvantage of using toluene
acidifies in partially filled vessel
Benzene is fast acting and is suited for
urgent biopsies
Disadvantages of using benzene
highly flammable, carcinogenic and can damage BM leading to aplastic anemia
Chloroform is used for
rough tissues (skin, fibroid, decalcified tissues) lymph nodes, nervous tissues and embryos
Chloroform is toxic to
liver
Disadvantage of using chloroform and its remedy
tissue will float , wrap in gauze
In using chloroform tissues do not become _______ and can lead to _____
translucent , wax deterioration
may be used for dense tissues like uterus
chloroform and cedarwood oil
Clearing time of cedarwood oil
2-3 days
Cedarwood oil can be used for
cytological studies like smooth muscles of skin and CNS tissues
It is recommended for embryos, insects and delicate specimens A. Chloroform B. Benzene C. Xylene D. Aniline oil
D
Tissues tend to become adulterated A. Chloroform B. Clove oil C. Xylene D. Aniline oil
B
Similar to chloroform but cheaper A. Chloroform B. Clove oil C. Carbon tetrachloride D. Aniline oil
C
extremely slow clearing agents and used only in double-embedding processes
Methyl benzoate, amyl acetate and methyl salicylate
can be used as xylene substitute
N-butyl acetate
obtained from citrus fruit
Limonene
Oil of bergamot and Oil of origanum is used to clear
smooth muscles of the skin
Oil of wintergreen is used for
delicate tissues
Carbon disulfide is used for
for smooth muscles; with foul odor
Carbon xylene is for
friable tissues
Terpineol is used for and has _______________ and __________.
eyes, has faint odor, low evaporation rate
good substitute for cedarwood oil
excellent clearing agent
High Test Aviation Lead Free gasoline-
Removal of dehydrating agent from tissues to replace it with a medium that will dissolve the wax with which the tissues to be impregnated A. Decalcification B. Fixation C. Dealcoholization/Clearing D. Embedding
C
Prolonged clearing time will cause
tissues to be brittle
The use of ____ will make tissues transparent and __________.
clearing, improve refractive index
Incomplete clearing will cause
uneven H&E staining
poor nuclear chromatin patterns
often used to assist in the selection of appropriate bone
specimens for processing.
Fine detail radiographs
should be used to prepare bone slices.
High-quality fine tooth saws
Bone specimens should be sawn into
thin slices
can cause considerable mechanical damage and force
bone fragments into the soft tissues present in the specimen.
Coarse saws
After fixation, depending on the amount of adjusted soft tissue, the toenail should be
rinsed off with soapy water once it becomes pliable
does not require any softening
Cartilage
three main types of decalcifying agents:
Those based on strong mineral acids
Those based on weaker organic acids
Those composed of chelating agents.
what happens if specimens are exposed for too long to acidic decalcifying agents
Nuclear and cytoplasmic detail are compromised
it will extract RNA and remove the purine and pyrimidine bases from DNA
effects of decalcifying agents on H&E staining can be reduced by
post-decalcification and removal, and by
appropriately adjusting the staining procedure.
In Formol-Nitric Acid, the yellow color imparted by nitrous acid formation may be prevented by
neutralizing the tissue with 5% sodium sulfate and washing in running tap water for at least 12 hours.
Addition of 0.1% urea to pure concentrated nitric acid will
also make discoloration disappear without considerably affecting the
efficiency of the decalcifying solution
In Phloroglucin-Nitric Acid, yellow color must be neutralized with
5% sodium sulfate and thoroughly washed with running tap water for at least 24 hours.
It does not require washing out before dehydration
Von Ebner’s Fluid
the only weak acid used extensively as a primary decalcifying
agent
Formic acid
-is a very weak decalcifying solution suitable only for
minute pieces of bone.
SULFUROUS ACID
pH of CITRIC ACID-CITRATE BUFFER SOLUTION
4.5
The rate at which EDTA will decalcify is pH
7.0
gives excellent results for soft-tissue
integrity, and best quality of both soft-tissue and hard-tissue staining
Neutral EDTA
EDTA works more rapidly at pH
10
The optimal pH when using EDTA
7-7.6
EDTA when used under pH 5 will result in
insolubility
EDTA when used over pH 8 will result in
tissue maceration
used to accelerate decalcification of trephine specimens for subsequent molecular analysis.
Sonication with EDTA
Due to the corrosive action of the acid, it is recommended that the thread be
be dipped in melted paraffin wax
In decalcification, what happens at 37°C
there will be impaired nuclear staining of Van Gieson’s stain for collagen fibers.
In decalcification, what happens at 55°C
the tissue will undergo complete digestion within 24-48 hours.
Acid decalcified tissues for frozen sections must be
washed in water or stored in formol-saline containing 15% sucrose or phosphate-buffered saline (PBS) with 15-20% sucrose at 4°C before freezing.
involves slow substitution of the water in the tissue with an organic solvent.
Dehydration
In dehydration, 85-95% alcohol will cause
considerable shrinkage and hardening of tissues leading to distortion
Another important role of the clearing agent is to remove a substantial amount of fat from the tissue which otherwise presents a barrier to wax infiltration.
True or False
True
used when the tissue is to be cleared directly from water, as in
a frozen section.
Glycerin and gum syrup
affects the speed of penetration of the clearing agent.
Viscosity
It is used for clearing, both for embedding and mounting procedures.
Xylene (Xylol)
Cedarwood oil becomes _______ upon prolonged storage and should be filtered before use.
milky
offer the clearing action with the lowest hazard rating of all xylene alternatives.
Orange oil based clearing agents
an efficient substitute for xylene, as it is non-hazardous, less expensive and causes less shrinkage of the tissue.
Coconut oil
The only drawback associated with coconut oil
tendency to get solidified at a lower temperature
