This document discusses tissue processing techniques. It describes how clearing agents act as intermediaries between dehydration and infiltration solutions. Xylene, toluene, and chloroform are common clearing agents that make tissues transparent. The criteria for choosing clearing agents and infiltration methods like paraffin wax embedding are also outlined. Automated tissue processors allow for standardized overnight processing through sequential alcohol, clearing, and infiltration solutions.

1. CLEARING
SUNIL KUMAR.P
St.john’s Medical College
Bangalore
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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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10. Infiltrating agents
• 1.paraffin 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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18. Automatic Tissue Processing Schedule
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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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