HISTOPATH: Clearing

de-alcoholization

other name for clearing

clearing

is the process whereby alcohol or a dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax.

translucent

appearance of the tissue when the solvent is entirely replaced

to facilitate the penetration of this embedding medium

why should clearing agents be miscible with paraffin wax?

to remove it from the tissue

why should clearing agents be miscible with alcohol?

xylene

dioxane

chloroform

cedarwood

most commonly used clearing agents for clearing in the embedding process

high refractive index

clearing agent will make microscopic tissue preparations transparent due to their _______.

xylene

most commonly used clearing agent for mounting sections

glycerin

gum syrup

are used when the tissue is to be cleared directly from water, as in a frozen section.

internal structures become visible to the naked eye

embryos and parasites become transparent upon clearing so that _______.

fast acting

miscible with alcohol

miscible with paraffin wax

miscible with canada balsam

should not evaporate easily

highlighted characteristics of a good clearing agent

low boiling point

clearing fluids with _______ are generally more readily replaced by melted paraffin

chloroform

a clearing agent which has a lower boiling point than xylene that takes longer to clear

viscosity

it also affects the speed of penetration of the clearing agent

xylene

most commonly used in histology laboratories

1/2-1 hour

clearing time for xylene

clearing

embedding

mounting

uses of xylene

xylene

suitable for most routine histologic processing

xylene

most rapid clearing agent

15-30 mins

clearing time of xylene for urgent biopsies

xylene

cedarwood

it does not extract out aniline dyes

xylene

it does not dissolve celloidin and can therefore be used for celloidin sections

nervous tissues

lymph nodes

tissues that are not suitable with xylene

incomplete dehydration of the tissue

cause of milky xylene

toluene

it may be used as a substitute for xylene and benzene

clearing

embedding

mounting

uses of toluene

1-2 hours

clearing time for toluene

toluene

a non carcinogenic clearing agent

toluene

it is relatively slower than xylene and benzene

toluene

tends to acidify in a partially filled vessel

toluene

highly concentrated solutions will emit fumes that are toxic upon prolonged exposure

benzene

preferred clearing agent for embedding process

15-60 mins

clearing time of benzene for urgent biopsies

benzene

it volatilizes rapidly in paraffin oven

benzene

it may damage the bone marrow

aplastic anemia

clinical manifestation caused by the toxicity of benzene

chloroform

thicker tissue blocks upto 1cm can be processed in this clearing agent

chloroform

this clearing agent does not make tissues translucent

6-24 hours

clearing time for chloroform

chloroform

it is recommended for tough tissues

skin

fibroid

decalcified tissues

tough tissues that are cleared by chloroform

tough tissues

nervous tissues

lymph nodes

embryos

tissues that are cleared by chloroform

chloroform

it is suitable for large tissue specimens

chloroform

a clearing agent that is not inflammable

chloroform

it is toxic to the liver

chloroform

it causes a relatively slow wax impregnation

chloroform

it does not make tissues transparent

chloroform

it is not very volatile in paraffin oven

chloroform

it can produce deterioration of the wax

chloroform

complete clearing is difficult to evaluate in this clearing agent

chloroform

cedarwood

tissues tend to float in these clearing agent

wrap the tissues with gauze

remedy for floating of tissues from the chloroform

chloroform

it evaporates quickly from a water bath

cedarwood

is used to clear both paraffin and celloidin sections

CNS

cytological studies

skin

smooth muscles

tissues recommended for cedarwood

oil wintergreen

oil bergamot

oils recommended for skin and smooth muscles

2

number of changes when using cedarwood oil

2-3 days

clearing time for cedarwood oil

chloroform

cedarwood

clearing agents recommended for dense fibrous tissues like uterus

5-6 days

clearing time of cedarwood for celloidin sections

cedarwood

this clearing agent often improves cutting of the sections

cedarwood

it is miscible with 96% alcohol

cedarwood

it is an extremely slow clearing agent

chloroform

cedarwood

clearing agents that are hard to remove

transfer the tissue from oil to benzene for 1/2 hour before placing the tissue in wax

how is wax impregantion hastened when cedarwood is present on the tissues?

cedarwood

quality is not always uniform and good in this clearing agent

superimposting absolute alcohol on the surface of the clearing agent

remedy for floating of tissues due to cedarwood oil

prolonged storage

cedarwood oil becomes mily due to _______

cedarwood

clearing agent that has been previously used to clear acetic-alcohol fixed tissues

35C

cedarwood oil cleraing acetic-alcohol fixed tissues may produce crystals with a melting point of approximately _______.

heat the solution to 200C

remedy for crystals formed by the cedarwood oil and restore its normal state

insects

embryos

very delicate spx

tissues cleared by aniline oil

aniline oil

it has the ability to clear 70% alcohol

clove oil

it has the tendency to become adulterated

clove oil

wax impregantion after clearing with this agent is slow and difficult

clove oil

tissues become brittle, aniline dyes are removed, and celloidin is dissolved in this clearing agent

carbon tetrachloride

its properties and disadvantages are similar to xylene/chloroform

amyl acetate

methyl benzoate

methyl salicylate

slow acting clearing agents

methyl benzoate

methyl salicylate

clearing agents used for double embedding techniques

carboxylol

dioxane

THF

clearing agents that can be used as both clearing and dehydrating agents

double embedding

it is the process when the tissues are infiltrated using celloidin and embedded using paraffin wax

celloidin

infiltrating medium used for double embedding

paraffin wax

embedding medium used for double embedding

paraffin wax

it is a routine medium used for both embedding and infiltration

skin

smooth muscles

tissues cleared by oil wintergreen and bergamot

xylene

benzene

clearing agents easily removed from from tissues

clove oil

it makes paraffin infiltration difficult and slower

limonene

it has a faint pleasant odor

citrus fruit/lemon

source of limonene

limonene

a clearing agent obtained from a citrus fruit

limonene

terpineol

it has a low evaporation rate

terpineol

it is a good substitite for cedarwood oil

terpineol

it is used for clearing eye spx

hydrocarbons

flammable liquids miscible with most organic solvents, paraffin wax, and coagulate nitrocellulose.

Leica Peloris

tissue processor that uses IPA rather than xylene as clearing agent

higher wax temperatures

needed for xylene free processing method to be successful

to eliminate IPA from tissues during infiltration

use of higher wax temp in a xylene free method of clearing

benzene

a far better transition solvent than xylene

toxic

carcinogenic

main drawbacks of benzene

chloroform

often used om dense tissues like uterus which can be cleared overnight without undie hardening