Histology TISSUE PROCESSING

TISSUE PROCESSING

4. TISSUE PROCESSING

Any treatment with a tissue that is required to impregnate it with a solidifying medium to

facilitate its fine sectioning for microscopic examination is named as tissue processing

which involves following four steps:

ƒ Completion of Fixation

ƒ Dehydration

ƒ Dealcoholization or Cleaning

ƒ Impregnation or Infiltration

4.1 COMPLETION OF FIXATION

In automatic tissues processors one or two jars of fixative are kept in the processing

schedule and processing is started from these jobs to ensure complete fixation.

4.2 DEHYDRATION

Tissues contain large amount of water, both intracellular and extracellular. This water must

be removed so that it may be replaced by wax. This process of water removal is called

dehydration. This process is done because the solidifying media, e.g. paraffin wax is not

miscible with water. Tissue biopsies contain two types of water; one coming from aqueous

fixative solution and secondly the water present inside the tissue as its component. 

In dehydration two mechanisms are involved as follows:

a) Hydrophilic Mechanism:

In this mechanism the dehydrating agent shows affinity for water and so attaches the water

from the tissue biopsy. This affinity is due to polar character of water molecules and

dehydrating agent used. 

b) Dilution Mechanism:

The dilution mechanism is very simple, i.e. when biopsies are transferred from one jar to

another the dehydrating agent dilutes out water and finally biopsies become completely free

from water.

Different types of dehydrating agents are being used in histopathology department. The best

agent is ethyl alcohol which has the advantage that it is not poisonous. Less common agent

are methanol, acetone, dioxane and isopropyl alcohol etc.

4.2.1 Ethanol:

Ethanol C2H5OH or ethyl alcohol is commonly called alcohol and obtained by the process

of fermentation of sugars which is a biochemical change brought about by certain microorganisms (bacteria and yeast). Initially complex sugars like sucrose and starch are

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glucose formed is then converted to ethyl alcohol by the action of an enzyme termed as

³=\PDVH´ which is also produced by micro organisms. The process is illustrated by the

following chemical reactions:

C12H22O11 invertase C6H12O6 + C6H12O6

C6H12O6 zymase 2C2H5OH + 2CO2

The ethanol (C2H6O) obtained in this way is RQO\  EHFDXVHWKHPLFUREHVFDQ¶WDFWDV

catalyst beyond this concentration. By fractional distillation ethanol of 95.6% concentration

LVREWDLQHGZKLFKLVWHUPHGDV³UHFWLILHGVSLULW´7RREWDLQDEVROXWHalcohol, the quicklime,

i.e. Cao is added which absorbs water and so after distillation absolute alcohol is obtained.

Properties of Ethanol:

Ethanol is an excellent dehydrating agent and shows a strong affinity for water. It is also

non-toxic but it is costly and bears a high excise duty. It is quicker in action but has a

hardening effect on tissue. So it is used in graded strengths, beginning from 70%, Transfer

of tissue directly from formalin to higher grades of alcohol, e.g. 85 or 95% is risky because

it will lead to distortion of tissues. In case of delicate tissue biopsies, e.g. brain and embryo, 

dehydration should be started with 50% alcohol.

Normally ethanol obtained by suppliers is not 100% and contains different qualities of

water, so poorly dehydrating the tissues. The purity is checked by using CuSO4 which exists

in 2 forms Anhydrous CuSO4 is white and when it absorbs water, hydrous form is obtained

which is blue in colour.

4.2.2 Methanol:

Methanol CH3OH is also known as wooden alcohol or wooden spirit and is obtained by

cracking a pyrolysis of wood at a temperature of 160o

C. Methanol is very toxic and

inflammable and has an unpleasant odor. It is rarely used as a dehydrating agent due to its

toxicity. 

4.2.3 Acetone:

It belongs to ketone family. It is cheap and quick in action as compared to ethanol but it

causes a lot of harness. Moreover, more volume is required for dehydration. It is highly

volatile and flammable so never be used in vicinity of open flame. It is not recommended

for automatic issue processors due to its volatility.It is possibly risky and there is

always a hazard of fire. It is used for quick dehydration in manual processing.

4.2.4 Dioxane:

Dioxane or diethylene dioxide is a unique reagent which has the unusual property of being

miscible with both water and molten paraffin wax. It is likewise miscible with liquor and

xylene. It is simple to use and produces little shrinkage. It is also very toxic and so no

longer recommended.

4.2.5 Iso Propanol:

If alcohol is not available isopropanol can be used. So it can act as a substitute for ethanol.

,W GRHVQ¶W KDYH DQ\ KDUGHQLQJ HIIHFW%XWODUJH YROXPHV are required and so takes a long

time for complete dehydration. Isopropanol is also termed as isoprophyl alcohol. 

[Ethylene glycol monoethyl ether or cellosolve can also act as dehydrating agents]

Box 5

The purchase and use of absolute ethanol is subject to many restrictions for customs and excise

purpose. Therefore, its substitute, i.e. 74o OP spirit (absolute industrial methylated spirit) is

normally used in laboratories. 3URRIVSLULWLVOHJDOO\GHILQHGDV³WKHVSLULWZKLFKDWWKHWHPSHUDWXUH

of 51o

F weighs exactly 12 ± 13th SDUWV RI DQ HTXDO YROXPH RI GLVWLOOHGZDWHU´$W o F it has a

specific gravity of 0.919 and contains 57.1% v/v or 49.2% w/w of ethanol. Spirits are described as

such countless degrees over evidence (Over powered) or under-confirmation (UP). Proof spirit is the standard and referred to as

100o

. A spirit stated as 70o would therefore be 30o UP (100o ± 70o

). A spirit stated as 160o would be

60o OP (100o + 60o

). 95% alcohol is equivalent to 60 OP, which means that 100 volumes of this

would contain as much ethanol as 166 volumes of proof spirit. 

As proof spirit (100) contains 57% ethanol, 74 OP (174) would contain:

57 X 174/100 % ethanol = 99%

100o

=57%, 1o

=57/100, 174o

=57/100x174 = 99.18%

4.3 CLEARING

The removal of dehydrating agent from a tissue biopsy is termed as cleaning or

dealcoholization. The chemicals used for the purpose are termed clearing agents. The term

clearing is used because it was found initially that most of the chemicals, e.g. xylene and

toluene increase the refractive index of tissue biopsy and make them transparent. However,

now a days it is proved that certain other chemicals, e.g. carbon tetrachloride and

chloroform have no such effects on biopsies but can be used to remove the dehydrating

agent. The term dealcoholization is also a misnomer because certain dehydrating agents are

not alcohols, e.g. acetone.

In histopathology clearing can also be used for the purpose of making tissues, embryos and

parasites transparent so that their internal structure is demonstrable to the naked eye. This is

done by impregnating them with a clearing agent whose refractive index is close to that of

the tissues themselves. For this purpose oil of wintergreen (methyl salicylate) and xylene

are used. 

Clearing Agents:

The clearing agents used must be miscible with both alcohol and paraffin wax. The most

common clearing agents are xylene, toluene, chloroform, cedar wood oil, carbon

tetrachloride, e.g., petrol.

4.3.1 Xylene:

It is an aromatic compound and benzene derivative. It is good clearing agent and increases

the transparency of tissue biopsies. It is comparatively cheaper and highly flammable.

Prolonged immersion of tissues (>3 hour) makes tissues hard and sectioning is difficult to

perform. It also causes brittleness of tissues. Tissue blocks 3mm in thickness are cleared in

15 ± 30 min but certain material, e.g. brain and lymph nodes become brittle if immersion is

prolonged. Such biopsies are best cleared by chloroform. When dehydration is not complete

and biopsy is kept in xylene jar, xylene become milky.

4.3.2 Toluene:

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harden the tissues. It is comparatively slower in action but somewhat expensive than

xylene. Its fumes may be toxic. It is flammable and has clearing property like xylene.

4.3.3 Benzene:

It is similar to xylene and toluene. Initially, benzene C6H6 was considered as a very good

clearing agent but now a days its use has been restricted due to its known carcinogenic

properties.

4.3.4 Chloroform:

It is recommended for nervous tissues, lymph nodes and embryos as it causes little

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Unlike Xylene and toluene, thick tissue biopsies upto 1cm can be cleared whereas in case of

xylene and toluene the thickness should not be more than 3 ± PP ,W GRHVQ¶W KDYHWKH

transparency effect on tissues. Traces of chloroform in tissues cause a problem in

sectioning. Its vapors are anesthetic and WR[LF2QKHDWLQJLWUHOHDVHDWR[LFJDV³3KRVJHQH´

Moreover, it may have a deleterious effect on the rubber sealing ring of the vacuum

impregnating bath.

4.3.5 Carbon Tetrachloride:

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transparency effect and just like chloroform releases phosgene gas.

4.3.6 Petrol:

Its properties are similar to xylene but are not recommended because different additives are

present in petrol and moreover it cannot be used as a clearing agent when methanol is used

as a dehydrating agent due to their non-miscibility.

4.3.7 Cedar Wood oil and Clove Oil:

These are rarely used as clearing agents because of their cost and slow action. So can be

used for delicate biopsies. But more time is consumed in clearing stage and later on in the

impregnation stage. It is due to high viscosity of oils. It is vital in research

labs and in embryological procedure. These oils impart consistency to tissues like skin and

dense fibrous material which facilitate subsequent sectioning.

These oils can tolerate small traces of water. This property is helpful in humid climate areas

where it is difficult to keep the dehydrating agent completely free of water. Cedar wood oil

for histological purpose is a thin, colourless, slightly yellow fluid distinct from the more

viscous type used for oil immersion objectives and which is unsuitable for clearing.