This document discusses the process of tissue processing, which involves fixing, dehydrating, clearing, and embedding tissue samples in paraffin wax to allow for thin sectioning. The key stages are fixation using chemicals like formalin to preserve tissue structure, dehydration using increasing concentrations of alcohol, clearing using solvents like xylene to make tissues transparent, and embedding in paraffin wax for sectioning. Automated and manual methods are described. Tissue microarrays allow evaluation of multiple tissue samples on a single slide by arranging small cores in a recipient paraffin block.

1. TISSUE PROCESSING
Presenter: Dr. Pranjal
Moderator: Dr. Pruthvi D.

2. OBJECTIVES
• Prerequisites
• Principle of Tissue Processing
• Stages of Tissue Processing
• Types of Tissue Processing
• Embedding
• Tissue Microarray

3. INTRODUCTION
Tissue processing is defined as the stepwise
treatment of tissues to remove all extractable
water from the tissue and impregnation of the
tissue with a solid support medium that is firm
enough to support it and give sufficient rigidity
to enable thin sections and that is soft enough not
to cause damage to the knife or tissue.

4. PRINCIPLE
A B C
Fixation Embedding
Dehydration,
clearing

5. FACTORS INFLUENCING RATE
• Specimen size
• Agitation
• Heat
• Viscosity
• Vacuum
• Ultrasonics.

6. PRE-REQUISITES
• Proper receipt and identification of the
specimen should be done.
• Proper sections of tissue should be taken, not
exceeding 3x2x0.4 cm.
• Proper labeling should be done of each section
of the tissue before putting it into the cassette.

7. STAGES OF TISSUE PROCESSING
• Fixation
• Dehydration
• Clearing
• Impregnation
• Embedding

8. FIXATION
• Process that preserves the morphological and
chemical characteristics of cells and tissues
and prevents autolytic and putrefactive
changes.
• It stabilizes and hardens the tissue with
minimal distortion of cells.

9. PROPERTIES OF FIXATIVES
• Cheap and easily available
• Stable and safe to handle
• Should cause fixation quickly
• Minimal damage to the tissue
• Even penetration of the tissue
• Retain the colour of the tissue

10. FIXATIVES
• Simple fixatives
• Compound fixatives

11. SIMPLE FIXATIVES
• Formalin
• Glutaraldehyde
• Osmium Tetroxide
• Potassium Dichromate
• Mercuric Chloride

12. COMPOUND FIXATIVES
• Formalin-based fixatives
• Mercurial fixatives
• Dichromate fixatives
• Picric acid fixatives

13. FORMALIN 10%
• Most commonly used fixative.
• Contains 10 ml of 40% formaldehyde with 90
ml of water.
• Reacts with proteins and forms cross-links
between the protein molecules.

14. ADVANTAGES
• Cheap, easy to prepare and stable.
• Tissue penetration is good.
• Best fixative for nervous tissue.
• Allows subsequent use of most staining
procedures including IHC.
• Natural tissue colour restored after fixation.

15. DISADVANTAGES
• Dermatitis
• Damage to nasal mucosa
• Formation of dark-brown formalin pigment
• May be carcinogenic
• Gradual loss of staining on prolonged fixation.

16. GLUTARALDEHYDE
• Aldehyde fixative with slower rate of diffusion
than formaldehyde but faster fixation.
• Mainly used for electron microscopy.
• Morphological features are well preserved.
• Best cross-linking agent for collagen.
• False PAS positivity.

17. MERCURIC CHLORIDE
Advantages:
• Excellent staining of nuclei and connective
tissues.
• Quick fixation.
• Staining of cytoplasm is more brilliant.
• Gives best results with metachromatic staining.
• Best preservation of details for photomicrography.

18. MERCURIC CHLORIDE
Disadvantages:
• Not recommended as fixative for demonstration of
nucleoproteins or sulfhydryl groups.
• Rate of penetration reduces after first few mm.
• Excessively hard and brittle tissue on prolonged
fixation.
• Corrodes all metals except Monel.
• Brownish mercurous chloride precipitate.

19. POTASSIUM DICHROMATE
Advantages:
• Used for fixation of mitochondria.
• No precipitation.
Disadvantages:
• Direct transfer to alcohol causes formation of
insoluble oxide. So, needs to be thoroughly washed in
running water.

20. DEHYDRATION
• Water in aqueous fixatives as well as in tissues are
immissible with paraffin wax and so has to be
removed.
• Dehydration is the process of removal of water and
fixative from the tissue.
• Achieved by immersing in increasing
grades/strengths of alcohol.

21. DEHYDRATING AGENTS
• Ethyl alcohol
• Methyl alcohol
• Isopropyl alcohol
• Butyl alcohol
• Tetrahydrofuran
• Acetone
• Dioxane

22. ETHYL ALCOHOL
• Fast-acting, clear, colorless, non-poisonous,
flammable and reliable liquid.
• Best dehydrating agent, produces total dehydration
and replaces aqueous fixative and unbound water.
• Dehydration should be complete.
• Dehydration is carried out using ascending grades of
concentrations.

23. Advantages Disadvantages
Non-toxic Hardening of tissue if left
for long periods
Graded alcohols starting
from 70% if used, avoids
shrinkage of tissues.
High excise duty, so
expensive and needs license
Ideal dehydrating agent for
delicate tissues
Requires 3-4 changes

24. METHYL ALCOHOL
• A clear, colorless and flammable liquid which is
miscible with water, ethanol and most organic
solvents.
• Highly toxic.

25. ISOPROPYL ALCOHOL
• Miscible with water, ethanol and most organic
solvents.
• Mainly used in microwave processing schedules.

26. Advantages Disadvantages
Easily available Expensive
Does not produce
overhardening or shrinkage
of the tissue
Cannot be used in celloidin
techniques
Fast acting, non-toxic and
reliable.

27. ACETONE
• Clear, colorless and flammable liquid
• Miscible with water, ethanol and most organic
solvents.

28. Advantages Disadvantages
Good, rapid dehydrating
agent
Highly volatile and
inflammable and shrinks the
tissue.
Cheap, readily available Tends to harden and
produce brittleness on
prolonged dehydration
Easily removed by most
clearing agents
Poor penetration and
removes lipids from tissue
during processing

29. Advantages Disadvantages
Mixes freely with water,
paraffin, xylene and alcohol
Expensive than alcohol and
odorous
Acts as both a fast
dehydrating and a clearing
agent
Has a cumulative toxic
action
Less shrinkage and
hardening of tissue
Areas of use should be well
ventilated
DIOXANE

30. TETRAHYDROFURAN (THF)
• 50% THF for 2 hours
• 100% THF for 3 changes of 2 hours each
• Equal parts of THF and wax for 2 hours
• Another 2 hours in wax.

31. CLEARING/DEALCOHOLIZATION
• Acts as an intermediary between the dehydration and
infiltration solutions.
• Should be miscible in both the dehydrating agent as
well as in the infiltrating solutions.
• When completely replaced by clearing agent, tissues
appear transparent, so this process is called clearing.

32. PROPERTIES OF A GOOD CLEARING AGENT
• Rapid or quick removal of dehydrating agent.
• Rapid penetration of tissues.
• Clear the tissue quickly without hardening or tissue
damage.
• Easily removed by molten paraffin wax.
• Low flammability, toxicity and cost.
• Does not evaporate too quickly in the wax baths.

33. CLEARING AGENTS
• Xylene
• Toluene
• Chloroform
• Benzene
• Carbon tetrachloride
• Cedar-wood oil

34. CLEARING AGENTS
• Citrus fruit oils
• Paraffin wax
• Histoclear
• CNP 30 and inhibisol
• Propylene oxide

35. XYLENE
• Flammable, colorless liquid with characteristic
petroleum odor.
• Miscible with most organic solvents and paraffin
wax.
• Most commonly used clearing agent in routine
histopathology.
• Refractive index is 1.50.

36. ADVANTAGES
• Rapid clearing agent
• Fairly cheap and easily available
• Makes the tissue transparent, end point of clearing
can be easily identified.

37. DISADVANTAGES
• Volatile and highly inflammable, vapor is an irritant.
• Prolonged treatment hardens the tissue and makes it
brittle.
• If xylene is added before completion of dehydration,
it becomes milky.
• Not advisable for brain and lymph nodes (too brittle).

38. TOLUENE
More volatile and flammable than xylene
Advantages:
• Does not harden tissue.
Disadvantages:
• Inflammable and potentially dangerous.
• Clears tissue less rapidly than xylene.

39. BENZENE
Advantages:
• Rapid action, little shrinkage, no hardening of tissues.
• Evaporates quickly.
Disadvantages:
• Highly flammable.
• Causes aplastic anemia and cancer in the laboratory
worker.

40. CHLOROFORM
Advantages:
• Tissue can be left longer without rendering them
brittle.
• Minimal shrinkage and hardening compared to xylene.
• Ideal for brain and lymph nodes.
• Nonflammable.

41. CHLOROFORM
Disadvantages:
• Highly toxic (phosgene).
• Highly expensive.
• Slow in its action (6-24 hrs).
• No change in refractive index, end point of clearing
cannot be made out.
• Anesthetic.

42. CITRUS FRUIT OILS
• Limonene reagents
• Derived from orange and lemon peels.
• Non-toxic and miscible with water.
• Can cause sensitization.
• Strong pungent odor.
• Oily and cannot be recycled.

43. CEDAR-WOOD OIL
Advantages
• No tissue shrinkage.
• Does not dissolve out aniline dyes.
• Good penetration.
• No damage or hardening of tissues.
• Best clearing agent.

44. CEDAR-WOOD OIL
Disadvantages
• Wax impregnation is slow.
• Highly expensive.
• Toxic and more viscous.

45. TECHNIQUE OF CLEARING BY
XYLENE
• Remove tissue from last beaker of alcohol.
• Place in 2 changes of xylene for 30 mins each.
• Transfer to paraffin wax.

46. INFILTRATION
• Permeation of the tissue with a support medium.
• Process of replacing the clearing agent by an
embedding medium.
• Helps in cutting thin sections easily.

47. EMBEDDING REAGENTS
• Paraffin wax
• Paraplast
• Paraplast plus
• Water soluble wax
• Polyester wax
• Micro-crystalline wax

48. EMBEDDING REAGENTS
• Resins
• Celloidin
• Agar
• Gelatin
• Carbowax

49. PARAFFIN WAX
• Most commonly used, cheap.
• White or colorless soft solid composed of long
straight-chained hydrocarbons.
• Permeates the tissue in liquid form, solidifies rapidly
at room temperature.
• No distortion of tissue, handled and stored with ease.

50. MELTING POINT OF PARAFFIN
• Defined as the temperature at which a drop of molten
wax becomes semisolid on the bulb of a slowly
rotating thermometer.
• Ranges from 47 to 64 degree celsius.
• Usually 58 to 60 degree celsius is preferred.

51. ADVANTAGES OF PARAFFIN
• Inexpensible.
• Provides sections of good quality.
• Easily adaptable to a variety of uses.
• Compatible with most routine and special stains.

52. PARAFFIN ADDITIVES
• Ceresin
• Bees wax
• Micro-crystalline wax
• Bayberry wax

53. PARAFFIN ADDITIVES- PURPOSE
• To cut thinner sections
• Increase hardness
• To get good ribbon sections.
• To alter the crystalline structure of wax to improve
sectioning.

54. OVENS/INCUBATORS
• Impregnation with paraffin wax takes place in an
incubator.
• Should have a temperature range of 50-60 degree
celcius.
• Should be large enough to accommodate an enamel
jar, one or two Coplin jars and few containers for wax
impregnation of tissues.

55. STEPS OF IMPREGNATION
• After blotting lightly with filter paper, tissue is
transferred into molten paraffin wax.
• Tissue should pass through at least 2 changes of wax.
• Impregnation with embedding media takes place in a
thermostatically controlled oven. Temperature
maintained at 2-3 degree celcius above the melting
point.
• Volume of wax: 25-30 times the volume of tissue.

56. DURATION OF IMPREGNATION
Depends on:-
• Size of tissue: Thicker tissue requires more time.
• Type of tissue: Dense tissue (bone, skin, CNS) require
more time.
• Clearing agent used: Xylene, toluene, benzene require
less time; Cedar-wood oil require more time and
several changes.
• Vacuum embedding oven: Reduces time required.

57. TYPES OF PARAFFIN WAX
• Liquid paraffin: used as oil for hematology, mounting
media for frozen section.
• Liquid paraffin with R.I. of 1.48: used for
spectroscopy.
• Petroleum jelly: Lubricant.
• Soft and white-melting point 56-60 degree celcius:
Histopathology.

58. ALTERNATES
• Paraplast
• Paraplast plus
• Water soluble waxes
• Resin
• Agar
• Celloidin
• Gelatin
• Plastic embedding media

59. PARAPLAST
• More elastic than normal paraffin wax (superior to
double embedding).
• Does not need cooling before cutting.
• Same melting point as routine paraffin wax.
• Represents a major technical advancement for routine
and research work.

60. PARAPLAST PLUS
• Contains DMSO (dimethyl sulfoxide).
• Allows more rapid penetration.
• Reduces the time for tissue processing.
• No need for filtering.

61. WATER SOLUBLE WAXES
• Polyethylene glycols with melting point of 38-40
degree celcius.
• Can be directly embedded from water.
• Used for demonstration of lipids and enzymes.

62. RESIN
• Embedding medium for electron microscopy
• Ultra-thin sectioning can be done.
• Undecalcified bone can be embedded.

63. AGAR
Used as a cohesive agent for small friable pieces of tissue
after fixation, a process called as double embedding.

64. CELLOIDIN
• Purified nitrocellulose.
• High resilience, hard or fragile tissues are more easily
cut.
• Heat is not needed during processing- minimal
shrinkage and distortion.
• Impregnation takes 2-3 weeks.

65. GELATIN
• Water-soluble wax.
• Used in sections of whole organs.

66. DOUBLE EMBEDDING
• Double embedding is a technique in which the tissue is
first impregnated with celloidin and subsequently
blocked in paraffin wax.
• Serial sections can be easily prepared.
• Extra degree of resilience is added.
• A tedious method.

67. TYPES OF TISSUE PROCESSING
• Hand processing
• Automated processing

68. AUTOMATED TISSUE PROCESSING
• Place the solution and paraffin in respective beakers.
• Timing leaver is set at 0.
• Baskets with the cassettes automatically change
position and takes a bath in different reagents kept in
different beakers in respective order.
• Casettes are opened next morning for embedding.

69. OVERNIGHT PROCESSING SCHEDULE
Sl. No. Reagents Duration
1. 10% Formalin I 1 hour
2. 10% Formalin II 1 hour
3. 50% alcohol/formalin 1 hour
4. 70% alcohol 1 hour
5. 95% alcohol I 1 hour
6. 95% alcohol II 1 hour
7. Absolute alcohol I 1 hour
8. Absolute alcohol II 1 hour
9. Xylene I 1 hour
10. Xylene II 1 hour
11. Paraffin I 2 hours
12. Paraffin II 2 hours

70. PREPARING PARAFFIN BLOCKS
• Leuckhart’s “L” molds
• Plastic embedding rings
• Plastic ice trays
• Paper boats
• Embedding trays
• Glass petridishes

71. LEUCKHART’S “L” MOLDS
L-shaped brass pieces placed in opposing positions and
can be manipulated to modify the size of block to be
prepared.

72. SECTION CUTTING
• Procedure of cutting and sectioning prepared blocks to
obtain thin strips of tissue.
• Instrument used is called a microtome.

73. TYPES OF MICROTOME
• Sliding
• Rotary
• Rocking
• Freezing
• Base sledge

74. TISSUE MICROARRAY
• Method used to evaluate numerous samples of tissue in
a short period of time.
• Multiple tissue samples can be arranged in a single
paraffin block using precision tools to prepare the
recipient block.

75. TECHNIQUE
• A hollow needle is used to take 100 or more tissue
core samples from specific areas of pre-existing
blocked tissue.
• Placed in a single array block.
• Sections are then taken from this block.
• Thus, a single slide containing hundreds of tissue cores
can be obtained.

76. USES
• IHC
• In-situ hybridization
• FISH
• Special stain control samples.
• Quality control sections for H&E.

77. ADVANTAGE
Cost-effective: only a small amount of reagent used per
slide.