Decalcification/Dehydration/Clearing Flashcards by LENNIE BELLE PORTENTO

Removal of calcium ions or lime salts from the organic extracellullar matrix, calcified collagen and surrounding tissues of bones.

Decalcification

How well did you know this?

Not at all

Perfectly

Ideal bone thickness?

1-3mm

How well did you know this?

Not at all

Perfectly

Decalcifying agent fluid to tissue ratio?

20:1

How well did you know this?

Not at all

Perfectly

What speeds up decalcification?

Heat and Agitation

How well did you know this?

Not at all

Perfectly

True or False:
More concentration acid solutions decalcify more rapidly but may destroy tissue

True

How well did you know this?

Not at all

Perfectly

Optimal temperature for decalcification?

Room temp

How well did you know this?

Not at all

Perfectly

The positively charged calcium ions are attracted to negative electrode from the decalcifying solution.

Electrolytic method

How well did you know this?

Not at all

Perfectly

Most rapid method. Physically dependent upon the supply of electric current for calcium removal

Electrolytic method

How well did you know this?

Not at all

Perfectly

Calcium is rapidly removed by the decalcifying soln contaning formic acid, increasing solubility from tissues

Ion Exchange Resin

How well did you know this?

Not at all

Perfectly

This increases solubility of tissues to facilitate removal of calcium.

Ammonium form of Polyklear resin

How well did you know this?

Not at all

Perfectly

What is the duration of Ion Exchange resin?

1-14 days

How well did you know this?

Not at all

Perfectly

This is a potent anticoagulant but a weak decalcifying agent.

EDTA

How well did you know this?

Not at all

Perfectly

Best decalcifier in EM and Immunohistochemistry

EDTA

How well did you know this?

Not at all

Perfectly

Inactivates alkaline phosphatase activity remedy?

Magnesium chloride

How well did you know this?

Not at all

Perfectly

EDTA specimen durations: small specimens, dense tissues

Small 1-3 weeks; Dense 6-8 weeks

How well did you know this?

Not at all

Perfectly

Most common method of decalcification

Use of Acid

How well did you know this?

Not at all

Perfectly

Most commonly used acid?

Nitric acid

How well did you know this?

Not at all

Perfectly

Acid used Routinely with a 24hr duration of process

10% Aqueous Nitric acid

How well did you know this?

Not at all

Perfectly

Nitric acid + Formaldehyde

What is its name and duration?

Formol Nitric acid; 1-3 days

How well did you know this?

Not at all

Perfectly

Chromic acid + ethyl alcohol.

What is its duration?

Perenyi’s Nitric acid; 2-7 days

How well did you know this?

Not at all

Perfectly

Double purpose; used as a decelcifying acid and tissue softener

Perenyi’s nitric acid

How well did you know this?

Not at all

Perfectly

Phloroglucin + nitric acid
Considered the most rapid

Phloroglucin Nitric

How well did you know this?

Not at all

Perfectly

Not routinely used; it is a weak and slow acid.

Used only for minute pieces of bone

Hydrochloric acid

How well did you know this?

Not at all

Perfectly

HCl +. NaCl

For teeth and small pieces of bone.

Surface decalcification of plaques

Von Ebner’s

How well did you know this?

Not at all

Perfectly

Recommended for decalcifying cartilage, respiratory spx, autopsy spx, bone marrow

Formic acid sodium citrate

These are weak and slow decalcifying agents

TCA, Sulfurous acid, (Hydrochloric acid)

Not commonly used acid because it is an environmental toxin and is highly corrosive on the skin

Chromic acid (Flemmings with HAC)

This acid contains chloroform as a preservative

Citric acid citrate buffer

What are the Methods of Decalcification?

- Electrolytic method - Ion Exchange method - Use of Chelating agents: EDTA - Use of Acid

Not a reliable method of testing completeness of decal. Done by bending, probing, pricking the tissue with needle

Physical/ Mechanical method

Most reliable mtd to test the completeness of decal. Can detect even the smallest amount of calcium.

X-ray/ Radiologic method

In this method, opaqueness = incomplete decalcification

X-ray/ Radiologic method

Disadvantage of Radiologic method?

Not suitable for Mercuric chloride fixed tissues

Cloudiness = to be decalcified further Clear = complete decalcification

Chemical method/ Calcium Oxalate test

State the process of Chemical method/ Calcium Oxalate test

5ml discarded fluid + strong ammonia —> check for alkalinity using litmus paper—> add ammonium oxalate —> stand for 30 mins

This test is carried out by adding calcium carbonate. Bubbles = incomplete decalcification

Bubble test

Decalcified tissues are neutralised by immersing in what?

Immersing in saturated lithium carbonate or 5-10% sodium bicarbonate

Most common method in neutralizing decalcified tissues

Rinsing in tap water

What is the third method for post decalcification?

Store in formol saline with 15% sucrose OR PBS with 15-20% sucrose at 4C

Tissue softeners for unduly hard tissues

- 1-2% HCl in 70% alcohol - Serenyi’s fluid - Molliflex - Lendrum’s method

Tissue softener, Lendrum’s method contain what

Spx in 4% phenol for 1-3 days

This uses increasing concentration of alcohol

Dehydration

Amount of dehydrating agent to tissue ratio

Should not be less than 10x the volume of the specimen

Initial concentration for Routine and Delicate tissues?

Routine 70-80% ; Delicate 30%

Routine dehydrating agent; fast acting and non-toxic

Ethanol

Dehydrating agent not commonly used; toxic Suited only for blood and tissue films

Methyl alcohol

Dehydrating agent Recommended for plant and animal microtechnique

Butyl alcohol

Ethanol and Xylene substitute; used in microwave technique

Isopropanol

This dehydrating agent dissolved paraffin

Pentanol

Prolonged storage in low concentration alcohol can _____________ tissues

Macerate

Prolonged storage in 70-80% can affect _______

Staining

Hasten dehydration by

Applying Heat at 37C

Accelerates dehydration and used as an Indicator for water saturation Blue discolouration = full saturation of dehydrating fluids with water

Anhydrous copper sulfate

Fixative and dehydrate tissues. Not commonly used. Evaporates early and is highly flammable

Acetone

Used for urgent biopsy and fast-acting

Acetone

Dehydrating and Clearing agent. Tissues tend to ribbon poorly.

Diethylene dioxide

Diethylene dioxide is also known as _________ Main disadvantage?

Dioxane; Extremely dangerous and toxic to man

3 changes of pure dioxane —> 3 changes of paraffin wax, embed in cool water

Graupner’s method

Tissue wrap in gauze bag and placed in a bottle with dioxane anhydrous calcium oxide or quicklime

Weisenberger’s method

In Weisenberger’s method, Dioxane ___________; anhydrous calcium oxide or quicklime _________________

Removes water; absorbs water removed by dioxane

Also known as cellosolve

Ethylene glycol monoethyl ether

Ethylene glycol monoethyl ether combustible at what temp

110-120F

Toxic by ingestion, inhalation, skin contact. Toxic to reproductive, fetal, urinary, and blood systems

Ethylene glycol monoethyl ether

Decomposes upon exposure to sunlight

Ethylene glycol monoethyl ether

Not commonly used; causes minimum shrinkage. For sections and smears

Triethyl phosphate

Can dehydrate and clear tissues Toxic when inhaled and ingested; has an offensive odor

Tetrahydrofuran

Vapors causes nausea, dizziness, and headache. May cause conjunctival irritation

Tetrahydrofuran

Removal of dehydrating agent from tissue to replace it with a medium that will dissolve the wax with wc the tissues is to be impregnated

Dealcoholization/Clearing

Routine clearing agent; Fast-acting at 30min-hours

Xylene

Clearing agent is not suited for Nervous tissues and lymph nodes. Incomplete dehydrated tissue turns this _______

Xylene; milky

Fast acting; suited for urgent biopsies. Highly flammable and carcinogenic, can damage BM leading to _________

Benzene; Aplastic anemia

This clearing agent is a substitute for Xylene and Benzene ______ Clearing time ________

Toluene 1-2 hours

This clearing agent acidifies in a partially filled vessel

Toluene

For rough tissues (/skin, fibroid, and decalcified tissues) for nervous tissues, lymph nodes, and embryos

Chloroform

Tissues in chloroform tend to float, remedy for this is ________

To wrap the tissue in gauze

Chloroform is toxic to what organ?

Liver

This clearing agent is for cytological studies like smooth muscles of skin and CNS tissues

Cedarwood oil

Cedarwood oil clearing time ___________

2-3 days; extremely slow clearing agent

Agent for clearing agent for embryos, insects, and delicate spx

Aniline oil

Agent where tissues tend to become adulterated

Clove oil

This clearing agent is similar to chloroform but cheaper

Carbon tetrachloride

Extremely slow agents used only in double embedding process

Methyl benzoate, Amyl acetate, Methyl salicylate

This clearing agent is a substitute for Xylene

N-butyl acetate

Clearing agent obtained from a citrus fruit

Limonene

Clearing agent for smooth muscles of the skin

Oil of bergamot Oil of origanum

Clearing agent for delicate tissues

Oil of wintergreen

Clearing agent for smooth muscles with foul odor

Carbon disulfide

Clearing agent for friable tissues

Carbon xylene

Clearing agent for eye specimens; with faint odor and low evaporation rate. Good substitute for Cedarwood oil

Terpineol

Excellent clearing agent

High Test Aviation Lead Free gasoline

These clearing agents may be used for dense tissues like the uterus

Chloroform and Cedarwood oil