Histopath Lab - Dehydration, Decalcification, clearing

1-3 mm

1-3 mm

It is the ideal thickness for decalcification

• Heat

• Agitation

Factors that speed up decalcification process

Electrolytic Method

This is the most rapid method of decalcification

Ion-exchange resin

• Calcium is rapidly removed by the decalcifying solution containing formic acid, thereby increasing solubility from tissues

• Process takes 1-14 days

EDTA

• Considered the best decalcifier in electron microscopy and immunohistochemistry

• Weak decalcifying agent in histopathology

• Inactivates alkaline phosphatase activity

1-3 weeks

EDTA for small specimens how long?

6-8 weeks

EDTA for dense tissues how long?

Addition of Magnesium fluoride

If EDTA inactivates alkaline phosphatase activity, how will you remedy it?

Nitric acid

The most commonly used acid decalcifier

10% Aqueous Nitric acid

• This acid is used for routine decalcification

• Process takes 24 hrs

• Nitric acid

• Formaldehyde

Composition of Formol nitric acid

1-3 days

Decalcifying duration of Formol nitric acid

Perenyi’s nitric acid

This is used as both decalcifying acid and tissue softener

Pholoroglucin Nitric acid

The most rapid decalcifying acid

Hydrochloric acid

• Weak and slow decalcifier

• Used only for minute pieces of bones

• Hydrochloric acid

• Sodium Chloride

Composition of Von Ebner’s solution

Von Ebner’s solution

This is used for teeth and small pieces of bones and for surface decalcification of blocks

Formic acid sodium citrate

• This is recommended for decalcifying cartilage

• Used for research and autopsy specimen, and BM

• Hydrochloric acid (HCl)

• Trichloroacetic acid (TCA)

• Sulfurous acid

Weak and slow decalcifying agents

Chromic acid / Flemmings with HAC

• It is considered as an environmental toxin

• It is highly corrosive on skin

Citric acid citrate buffer

It contains chloroform as preservative

Physical Method

• Not a reliable method but easy to do

• Done by tissue bending or pricking/probing tissue with needle

X-ray Method

• Considered the most reliable/accurate since it can detect even the smallest amount of calcium

• Presence of opaqueness must be observed since its presence signifies incompleteness of decalcification

• NOT SUITED for Mercuric chloride-fixed tissues

Chemical Method (Calcium Oxalate Test)

• Transfer 5mL of discarded fluid in a tube

• Turn it alkaline by adding strong ammonia to the tube

• Use litmus paper to check if it is alkaline

Bubble Test

• This test is carried out by adding Calcium carbonate

• Presence of bubbles mean decalcification process is not complete

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

Decalcified tissues are neutralized by:

• 1 or 2% HCl in 70% alcohol

• Perenyi’s Fluid

• Molliflex

Tissue softeners for unduly hard tissues:

Lendrum’s Method

This method involves immersing specimens in 4% aqueous phenol for 1-3 days

4% aqueous phenol

What is the solution used in Lendrum’s Method

Dehydration

This process is done after fixation and prior to wax impregnation

10x

Amount of dehydrating agent volume should not be less than ______ the volume of specimen

It will macerate tissues

Use of low concentration dehydrating agent will do what to tissues?

It will harden ONLY the surface of tissues

Use of high concentration dehydrating agent will do what to tissues?

70-80%

What is the initial concentration for routine dehydration?

30%

What is the initial concentration for delicate tissue dehydration?

Ethanol

• This is the routine dehydrating agent

• It is fast acting and non-toxic

Methyl alcohol

This alcohol is toxic and suited only for blood and tissue films

Butyl alcohol (Butanol)

This alcohol is used for plant and animal micro-technique

Isopropanol (IPA)

• It is used as an ethanol substitute

• It is used for microwave technique

• It can be a saline substitute in process of clearing

Pentanol

This alcohol is used for dissolving paraffin

FALSE

TRUE OR FALSE

The use of alcohol agents should be in descending concentration

Anhydrous copper sulfate

• This will accelerate dehydration process and can be used as an indicator for water saturation

• Blue discoloration of this will indicate full saturation of dehydrating fluid with water

Acetone

• It can fix and dehydrate tissues at the same time

• It evaporates easily and is flammable

• Fast acting. for urgent biopsies

Diethylene dioxide

• It is also known as Dioxane

• It can act both as a dehydrating and clearing agent

• Tissues tend to ribbon poorly with this agent

• Extremely dangerous, poisonous to humans

Graupner’s Method

This method of Dioxane dehydration involves 3 changes of pure dioxane followed by 3 changes of paraffin wax, embed the cool in water

Weiseberger’s Method

• This method of Dioxane dehydration involves tissue wrapped in a gauze bag and placed in a bottle with dioxane and anhydrous calcium oxide or quicklime

• NOTE: Dioxane will remove the water

Ethylene glycol monoethyl ether

• This is also known as Cellosolve

• It is combustible at 110-120 degrees Fahrenheit

• It decomposes when exposed to sunlight

Cellosolve

Ethylene glycol monoethyl ether is also known as?

Dioxane

Diethylene dioxide is also known as?

Triethyl phosphate

• This is recommended for dehydrating sections and smears

• It causes minimum shrinkage

Tetrahydrofuran (THF)

• It can dehydrate and clear tissues at the same time

• Toxic when ingested or inhaled

• It has an OFFENSIVE ODOR

• It may cause conjunctival irritation

Xylene

• This is the routine clearing agent

• It has a rapid clearing time of 30 mins to a few hrs

• It becomes milky when an incompletely dehydrate tissue is immersed in it

Toluene

• This is used as a substitute for Xylene and Benzene

• Clearing time is 1-2 hrs

• It acidifies in a partially filled vessel

Benzene

• This clearing agent is suited for urgent biopsies

• It is highly flammable and carcinogenic, can damage BM leading to aplastic anemia

Chloroform

• This clearing agent is used for rough tissues, nervous tissues, lymph nodes and embryos

• Tissues tend to float with this agent

• It is highly toxic to the liver

• Tissues do not become translucent with this agent

• Use of this agent leads to wax deterioration

• Chloroform

• Cedarwood oil

These clearing agents are used for dense tissues like the uterus

Cedarwood oil

• Clearing time for this agent is 2-3 days

• It is used for cytological studies like smooth muscles and CNS tissues

• Becomes Milky in Prolonged Storage

Aniline oil

It is recommended for clearing embryos, insects, and delicate spx.

Clove oil

Tissues tend to become adulterated with this clearing agent

Carbon tetrachloride

It is similar to chloroform but is cheaper

Methyl benzoate

This clearing agent is used only in a double-embedding process

N-butyl acetate

It can be used as a Xylene substitute

Limonene

Clearing agent obtained from a citrus fruit

• Oil of bergamot

• Oil of origanum

Both of these oils can be used to clear smooth muscle of the skin

Oil of wintergreen

This oil is used for delicate tissues

Carbon disulfide

This clearing agent is used for smooth muscles

It has a FOUL ODOR

Carbon xylene

This clearing agent is used for friable tissues

Terpineol

• This clearing agent is used for the eyes

• It has a FAINT ODOR

• It has a low evaporation rate

• Good substitute for cedarwood oil

High Test Aviation Lead Free gasoline

An excellent clearing agent