MODULE 2 - Decalcification & Dehydration/Clearing Flashcards by SHAINA MAE CASPE

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

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Both decalcification and processing depend on

Bone thickness

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Ideal thickness of bone tissues

1-3 mm

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Decalcification must be done following

Fixation

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Ratio of fluid to tissue in decalcification

20:1 or 10-20x the volume of tissue

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Factors that can speed up decalcification

Heat and agitation

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This can decalcify more rapidly but may destroy tissue.

Concentrated acid solutions

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Optimal temperature in decalcification

Room temperature

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Decalcification can be done on autotechnicon

True or false

False, it must be done MANUALLY

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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

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It Inactivates alkaline phosphatase activity

Use of chelating agent: EDTA

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A combination of nitric acid and formaldehyde

Formol nitric acid

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It requires fumehood.

Formol nitric acid

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Duration of ion exchange resin

1-14 days

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Use of chelating agent: EDTA is best for

EM and immunohistochemistry

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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

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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

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Duration for small specimen in using chelating agent: EDTA

1-3 weeks

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Duration for dense tissue in using chelating agent: EDTA

6-8 weeks

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Duration in using Ion Exchange resin.

1-14 days

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Use of Chelating agent: EDTA inactivates alkaline phosphatase activity, what is the remedy?

Add magnesium chloride

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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

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The most rapid method of decalcification
A. Ion exchange resin
B. Electrolytic method
C. Use of Acid
D. Use of chelating agent: EDTA

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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

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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 ```

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 ```

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 ```

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 ```

``` 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 ```

``` It is not commonly used because it is toxic. A. Ethanol B. Methyl alcohol C. Butyl alcohol D. Pentanol ```

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 ```

``` It dissolve paraffin. A. Ethanol B. Methyl alcohol C. Butyl alcohol D. Pentanol ```

Enumerate dehydrating agents under alcohol

1. Ethanol 2. Methyl alcohol 3. Butyl alcohol/butanol 4. Isopropanol/IPA/isopropyl alcohol 5. Tertiary butanol 6. 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

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 ```

``` Tissues tend to become adulterated A. Chloroform B. Clove oil C. Xylene D. Aniline oil ```

``` Similar to chloroform but cheaper A. Chloroform B. Clove oil C. Carbon tetrachloride D. Aniline oil ```

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 ```

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

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