2. INTRODUCTION
• Clearing reagents acts as an intermediary
between the dehydration and infiltration
solutions.
• They should be Misible with both solutions.
• Most clearants are hydrocarbons with refractive
indicies similar to protein.
• When the dehydrating agent has been entirely
replaced by most of these solvents the tissue has
a translucent appearance ; hence the term “
clearing agent”.
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3. DEFINITION
• The term “Clearing “ relates to the clear and
translucent appearance of the tissue after
treatment with the clearing agent.
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4. Criteria for choosing a suitable
clearing agent are
• 1. Rapid penetration of tissue
• 2.Rapid removal of dehydrating agent
• 3.Ease of removal by melted paraffin wax
• 4.Minimal tissue damage
• 5.Low flammability
• 6.Low toxicity
• 7.Low cost
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5. Clearing agents suitable for routine use
(TYPES)
• 1.Xylene
• 2.Toluene
• 3.Chloroform
• 4.Benzene
• 5.Cedar wood oil
• 6.Paraffin oil
• 7.Citrus fruit oils
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6. XYLENE
• Xylene is a flammable
• Color less liquid
• Petroleum or aromatic odour
• Misible with most organic solvents and paraffin
wax.
• It is suitable for clearing blocks less than 5 mm in
thickness and rapidly replace the alcohol from
the tissue.
• Over exposure to Xylene causes hardening of
tissues.
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7. TOLUENE
• This has similar properties of Xylene but
causes less brittleness.
• It is more flammable and volatile than xylene
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8. CHLOROFORM
• Chloroform is slower in action than xylene but
causes less brittleness.
• Thicker tissue blocks can be processed, greater
than 1 mm in thickness.
• It is non-flammable but highly toxic
• Most commonly used for CNS
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9. Infiltration or Impregnation
• After clearing the tissue is transferred into a
chamber containing molten paraffin wax.
• During this stage………….
• the clearing agent is eliminated from the
tissue by diffusion into the molten wax and
the wax in turn diffuses into the tissue.
• The tissue should pass at least two changes of
wax to achieve proper impregnation by wax.
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11. Other alternative infiltrating and
embedding agents
• 1.Resin
• 2.Agar
• 3.Gelatin
• 4.Cellodin
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12. PARAFFIN WAX
• Paraffin wax is the most commonly used
infiltrating and embedding medium in
histopathology.
• Paraffin wax is a mixture of long chained
hydrocarbons produced in the cracking of mineral
oil.
• The waxes commonly used have a melting point
ranging between 50 – 60oC.
• The temp. of the wax can be maintained using a
thermostatically controlled oven.
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13. • The average time taken for complete
impregnation of a piece of tissue 5 mm thick is
about 3 hours.
• But dense and thick tissues require longer
time.
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14. Tissue processing
• Methods : Manual
• Automated Method by
• (Automatic tissue processor)
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15. Tissue processing by Using Automatic
Tissue Processor
• Requirements
• 1. Alcohol solutions of various con% such as
• a.70 %
• b.95%
• c.100% ( absolute Alcohol)
• 2. Xylene or Acetone
• 3.Chloroform
• 4.Parafffin
• 5.Automatic tissue processor
• 6.Tissue
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16. Procedure
• 1. Place the solutions and paraffin in
respective beakers of the equipment
• 2.The timing level is set at Zero and the
machine is started (overnight Processing)
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17. conti…….
• 3.The basket with the cassettes automatically
changes position and takes a bath in different
reagents kept in the beakers in order to
accomplish
• - Dehydration
• - Clearing
• - Infiltration
• - The final dip in the warm paraffin
• 4.Cassettes are opened next day morning for
embedding.
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19. Advantages of Automatic Tissue
processing
• 1.Custom programs specific to tissues being
processed, addition of vaccum, agitation or
heat at any stage.
• 2.Rapid schedules.
• 3.Fluid and fume containment
• 4.Environmentally friendly reagents
• 5.Time delay for start of processing schedules
• 6.Reagent management
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20. Maintenance & care Of Automated
Tissue Processor
• 1.Any spillage or overflow should be cleaned
immediately.
• 2.Accumulation of wax on any surface should
be removed.
• 3.The temp. of the paraffin wax bath should
be set to 3oC above the melting point of wax.
• 4.Timings should be checked when placing the
cassettes in the processor
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21. Quality control ….. For Automatic
tissue processor
• Temp. of all paraffin wax dispensers,
• flotation water baths and automated
processors are carefully monitored.
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