tissue processing introduction basics ppt

CONTENTS
 INTRODUCTION
 HISTORY
 SPECIMEN TRACKING
 TISSUE PROCESSING
 STAGES OF TISSUE PROCESSING
 EMBEDDING
 FACTORS INFLUENCING PROCESSING
 TROUBLE SHOOTER
 TISSUE PROCESSING IN SPECIAL STUDIES

TISSUE PROCESSING
 Stepwise treatment of the tissue aimed to
remove all extractable water & embed
in solid medium firm enough to support &
give rigidity to cut thin sections without
damaging the tissues.

HISTORY
 MARIE FRANCOIS
BICHAT:
gross dissection of
tissue samples

 IN 1666, ROBERT
BOYLE : reported
that
Immersion of tissue
in spirit of wine
prevents post mortem
decay.
 preserves natural
state of tissue.
Hardens tissue.

 IN 1840, ADOLPH
HANNOVER:
 Chromate of potash.
 Prevent fermentation
& putrefaction.
 Hardens tissue.

 1850, FRANZE
SCHULZE:
Osmic acid(fixative).
 1859,
ALEXANDER
BUTLEROV:
Formaldehyde
(fixative).

 1869, EDWIN
KLEBS:
Paraffin wax

SPECIMEN TRACKING
 All tissue received in the laboratory is given a
unique number.
 NUMBERING PATTERN:
 Chronological order of the specimen.
 Oblique sign (/)
 Last two numbers of the year.
eg: 1/21.
A 1/21
 Entered in register book.

SELECTION OF TISSUE
 Adequately fixed.
 A brief gross description of the specimen.
 Small specimen, entire tissue is processed.
 Big specimen, based on specimen received
representative bits given.
 Max surface area 3×2cm.
 Not be more than 4mm thick (2-3mm).

tissue processing introduction basics ppt

 1% EOSIN:
 Added to small specimen to visualization
during embedding.
 30 mins prior to processing
 Remain during processing & washed of
before staining

 Each capsule is
labelled with
alphabet & number
assigned to the
specimen in a small
paper using graphite
pencil.
 Tissue bit along with
designated number
is kept in cassette (
or capsule) & lid is
closed.

CASSETTES/ TISSUE CONTAINER
 Cassettes have
perforation which
allow free access of
the tissue to different
solutions used in
processing.
 Tissue fragments
may shrink during
processing, hence
appropriate capsules
selected or wrapped
in a filter paper.

STAGES OF TISSUE PROCESSING
1. Fixation
2. Dehydration
3. Clearing
4. Impregnation
5. Embedding

EVOLUTION OF TISSUE PROCESSING.
 MANUAL(HAND PROCESSING).
 AUTOMATED ENCLOSED
 CAROUSEL TYPE/ TISSUE TRANSFER.
 STATIONARY CHAMBER PROCESSOR/ FLUID
TRANSFER.
 MICROWAVE TISSUE PROCESSOR.
 TISSUE-TEK XPRESS

MANUAL PROCESSING
 8 hours • Affordable • Time flexibility
SCHEDULE:
1.Dehydration
 95% alcohol- 2 changes of 1 hour each .
 Acetone- 2 changes of 1 hour each
2.Clearing Chloroform- 2 changes of 1 hour each
3. Impregnation Paraffin- 2 changes of 1 hour
each
( Volume of the fluids taken is 10 times the
tissue volume)

1.Formalin - 2 changes of 2 hours
2.Dehydration
 70% ethanol - 2 hours.
 80% ethanol - 2 hours.
 90% ethanol - 2 hours .
 Absolute alcohol- 2 changes ,1 hour each.
3. Clearing Xylene - 3 changes of 45 minutes
each.
4.Impregnation Paraffin wax - 2 changes of 1
hour each.
( Volume of the fluids taken is 10 times the
tissue volume)

MICRO WAVE
 Invented by
PERCY SPENCER
.
 Heat picks up
diffusion of liquids
in & out of tissue.

PROCESSING SCHEDULE
 DEHYDRATION: ethanol at 67 degree
centigrade for 5 minute.
 CLEARING: Isopropyl alcohol at 74 degree
centigrade for 3minutes.
 IMPREGNATION: paraffin at 65 degree
centigrade for 2 min.
 Followed by 80 degree centigrade for 5 min

FIXATION:
 Prevent autolysis by denaturing the proteins &
maintain cellular structure.
 Most commonly used: 10% NEUTRAL
BUFFERED FORMALIN.

POST FIXATION TREATMENT
 PICRIC ACID FIXATIVES ( BOUIN’S):
 Cassettes are directly placed in 70% of
alcohol.
 ALCOHOL FIXATIVES( CARNOY’S
FLUID):
 Directly placed in 100% alcohol.

DEHYDRATION
 Removes cellular water & residual fixative.
 Water in tissue is found in two forms:
1. Free form
2. Bound form(integral part of macromolecules
of the cells).
 Only free form to be removed by using graded
alcohols.
 Total time required depends on volume, type
of tissue & dehydrating agents.

 Graded concentration of alcohol:
1. For all specimen: 70% , 95% & 100%.
2. Delicate tissue: 50%

DEHYDRATING AGENTS:
1. Ethyl alcohol.
2. Methyl alcohol.
3. Isopropyl alcohol.
4. Acetone.
5. Dioxane.
 Dehydrating agents should not be less than
10 times the volume of tissue.

ETHANOL ADVANTAGES DISADVANTAGES
Hydrophilic,
miscible with
other organic
solvent
Flammable
Non- toxic Expensive
Reagent of
choice in
electron
microscopy
Requires 3-4
changes

METHANOL ADVANTAGES DISADVANTAGES
Miscible with
other solvents
Volatile
Flammable
Highly toxic
ISOPROPYL
ALCOHOL
Microwave
processing.
Expensive
No shrinkage or
over hardening.
Cannot be used
in celloidin
techniques
Fast acting,
reliable & non-
toxic
Impair staining
with eosin

ACETONE
Cheap Volatile
Quick action
compared to
alcohol
Hard & brittle
tissue
Manual
processing
DIOXANE
Fast
dehydrating,
clearing agent
expensive
Less shrinkage
& hardening of
tissue
toxic
Mix freely with
paraffin, water,

UNIVERSAL SOLVENTS
 Both properties of dehydrating & clearing.
 Hazardous in nature, not used routinely
 Eg: Dioxane, tertiary butanol,
tetrahydrofuran

CLEARING( DEALCOHOLIZATION)
 Displaces dehydrating solutions, making tissue
components receptive to infiltrating medium.
 Also dissolve lipids , impede the wax
penetration.

PROPERTIES OF GOOD CLEARING
AGENT
1. Rapid penetration of tissue.
2. Rapid removal of dehydrating agents.
3. Clear the tissue quickly without hardening or
tissue damage.
4. Easily removed by melted paraffin wax.
5. Low flammability & cost.
6. Not evaporate too quickly in the wax baths.

CLEARING AGENTS ADVANTAGES DISADVANTAGES
Xylene  Rapidly acting
 End point of
clearing can be
determined
 Affordable
 Flammable
 Makes tissue
brittle, hard
 Unsuitable for
brain, lymph
node
Toluene  Same as xylene
 Does not
harden tissue on
prolonged
immersion
 Similar to xylene
 Slower

Chloroform Manual method.
Less hardness,
brittleness even
on prolonged
immersion.
 good clearent
for neural tissue
& lymph node.
 slow.
Toxic
(phosphogene ).
Benzene  rapid.
Minimal
shrinkage.
CARCINOGENI
C.
Inflammable.
Methyl salicyclate  good for
delicate tissue
Does not mix
well with paraffin.

Clove oil  Less
shrinkage
 Causes
brittleness.
 Slow.
Dissolves
celloidin.
Cedar wood oil  Least
hardening effect.
Good for
delicate tissues
like embryos &
dense fibrous
tissue.
 Slow.
 Expensive
Toxic
Oil of origanum  Does not
dissolve celloidin
 Expensive

INFILTRATION / WAX IMPREGNATION
 Permeation of the tissue with a support medium.
 Improve optical differentiation.
 Adequate consistency to ensure good sectioning.
 Process of replacement of clearing agent by
an embedding medium.
 Volume- 25 times the volume of tissue

IMPREGNATING AGENTS
1. Paraffin wax
2. Paraplast
3. Embeddol
4. Bioloid
5. Carbowax

1. PARAFFIN WAX
 Introduced by EDWIN KLEBS in 1869.
 Inexpensive.
 Most commonly used.
 Provides sections of good quality.
 Easily adaptable & compatible with most
routine & special stains, immunohistochemistry
protocols.

◦ Mixture of long straight chained hydrocarbons
produced in the cracking of mineral oil.
◦ Solid at room temperature, allows sectioning
of 3-4microns in thickness.
◦ Wide range of melting point (47-64 degree
Celsius).
◦ Low melting point: softer paraffin, for
impregnation.
◦ High melting point: harder paraffin, for
embedding.

PARAFFIN WAX ADDITIVES
 Modify consistency, melting point & increase
hardness to cut thinner sections.
 To increase the stickiness to get good ribbon
sections.
 ADDITIVES USED:
1. Ceresin: derived from Ozokerite
2. Microcrystalline wax
3. Bees wax
4. Bayberry wax
5. Dental wax

2. PARAPLAST
 Mixture of highly purified paraffin wax &
several synthetic polymers.
• Melting point is 56-57 degrees.
Advantages : –
• No need for rapid cooling
• Easy ribboning.
• Large blocks & dense bone
blocks can be cut with ease

3. EMBEDDOL:
 Synthetic wax substitute similar to paraplast.
 Melting point: 56-58 degree Celsius.
 Less brittle & compressible.
4. BIOLOID:
 Semisynthetic wax
 Used for embedding eyes

5. CARBOWAX:
 water soluble wax, embedding can be done directly after
fixation.
ADVANTAGES DISADVANTAGES
1. Less time for processing. 1. Highly HYGROSCOPIC-
blocks & unstained sections
must be kept dry.
2. Dehydrating & clearing
agents not required.
2. Difficult to obtain good
sections.
3. Does not remove lipids.
4. Good for enzyme
histochemical studies.
5. Less shrinkage & distortion
of tissue.

EMBEDDING OR BLOCKING
 Orientation of the tissue sample in a
support medium to create a tissue
“ block” suitable for sectioning.

EMBEDDING CENTRES:
1. HEAT STORAGE
CHAMBER
2. PARAFFIN WAX
RESERVOIR
3. PARAFFIN WAX
DISPENSER
4. EMBEDDING
AREA

5. COLD PLATE:
 -5 to +5 degree
Celsius.
 Around 60 blocks
can be cooled.
 Assists in
solidification of
blocks.
6. DRAIN TRAY: for
excess wax.

EMBEDDING MOULDS
1. LEUCKHART’S
“L” MOULDS:
 ‘L’ shaped pieces
of heavy metal.
 placed opposite to
each other on a flat
metal or glass
surface to form
moulds of
desirable sizes.

2. TISSUE TEK SYSTEM 1
 Select appropriate
sized mold
 Place the tissue.
 Plastic embedding
ring is placed on
top of the base
mold
 Molten wax is
poured in upto the
top of plastic ring
 The wax is cooled

ADVANTAGES
DISADVANTAGES
 Block can be cut
without trimming
 Wax filled plastic
embedding ring
functions as the block
holder, eliminates
the step of mounting
paraffin block on a
block holder.
 No de-blocking
needed & makes
recutting of tissues
 Space needed for
filling & storing the
blocks is almost
double.
 Expensive

3.TISSUE TEK SYSTEM 2
 Modification of the
plastic embedding
ring.
 The plastic
embedding
cassette has1.5 X
4mm sloping to
3mm at top & 5mm
deep.
 Base of
embedding
cassette has one

ADVANTAGES
DISADVANTAGES
 Requires 1/3rd less
wax than plastic
embedding rings
 less storage space
 Shallow cassette
is used, so thin
sections to be
taken for
processing.

4. PEEL- A-WAY :
disposable thin
plastic embedding
molds. No trimming

ALTERNATIVE EMBEDDING
MEDIA
1. Celloidin technique
2. Gelatin technique
3. Synthetic resin
4. Paraplast plus
5. Resin
6. Agar.

1. CELLOIDIN EMBEDDING: Purified
collodion (mixture of di
&trinitrocellulose).
METHODS DRY METHOD WET METHOD
USES • Eyes • bones, teeth,
whole organs &
large brain blocks.
TECHNIQUES • Use of cedar wood
oil & chloroform
instead of alcohol
for treating the
blocks.
• block & sections
are stored in 80%
alcohol.
• during cutting
both knife & block
to be kept moist
with alcohol.

• Excellent penetration. • Slow, highly
inflammable.
•Avoids damaging effects
of heat
• Exhaustive technical
expertise.
•Difficult to obtain
ribboning.
•Difficult to section <
10microns tissue bit.

2. GELATIN EMBEDDING
 Water soluble wax.
 Used in production of whole organs using
GOUGH- WENTWORTH TECHNIQUE, in
frozen section of friable or partially necrotic
tissue or small fragments.
 Block is immersed in 10% of formalin after
embedding to convert gelatin into irreversible
gel.

1. Dehydration & clearing
not needed.
1. Holds the stain, giving
an indifferent background
to the section.
2. Prevents distortion of
tissue.
3. Good for tissue like
endometrial curettage,
lung, spleen, pancreas &
friable tissue.

3. SYNTHETIC RESINS
 USES:
1. Ultrastructural studies.
2. Hard tissues & implants.
3. High- resolution light microscopy.
 TYPES:
 ACRYLIC RESINS: acrylates, methacrylates
 EPOXY RESINS: araldite, glycerol, cyclohexene
dioxide.
ADVANTAGES: hard blocks permitting very thin
sections( 0.5-2 microns).
DISADVANTAGES: expensive, contact dermatitis &
special microtomes are required.

4. PARAPLAST PLUS:
 Contain DIMETHYL SULFOXIDE.
 Rapid penetration.
5. Resin:
used as embedding media in:
 Electron microscopy.
 Ultra thin section for high resolution.
 Undecalcified bone.
6. AGAR:
 Used as a cohesive agent for small friable pieces of
tissue after fixation.

ORIENTATION OF TISSUES:
 Demonstration of proper morphology.
 Tissue should offer least resistant against microtome
blade during cutting.
TISSUES REQUIRING SPECIAL ORIENTATION:
1. SKIN BIOPSY: • cut at right angles to
the surface, full
thickness must be
visible.
2. TUBULAR
STRUCTURES:
• cut at right angles ,
lumen can be clearly
visible

3. EPITHELIAL BIOPSY: • Cut at right angles to
the surface.
•Epithelial surface
should cut last,
minimize compression
& distortion of epithelial
layer.
4. MUSCLE BIOPSY: • embedded in such a way
that both longitudinal &
transverse section can be
obtained.
5. BONE MARROW &
TRUE CUT BIOPSY:
• Placed horizontally.

6. MULTIPLE PIECES
OF A TISSUE:
• Oriented side by side
with epithelial surface
placed in same direction.
7. INKED TISSUE OR
MARGINS:
• Inked surface placing
downwards

TISSUE MARKING
 Should be
insoluble.
 Not penetrate the
tissue.
 Not react with
histological stains.
 Clearly identifiable
both grossly &
microscopically.
 Eg: indian ink,
silver nitrate &
alcian blue

DOUBLE EMBEDDING
 tissue are first impregnated with celloidin &
subsequently blocked in paraffin wax.
ADVANTAGES DISADVANTAGE
1. Good support.
2. Good ribboning.
1. Tedious method

RE- EMBEDDING
 Orientation of the tissue is improper.
 Melted in oven.
 Tissue is properly oriented using new paraffin
block

FACTORS INFLUENCING RATE OF PROCESSING
1.Specimen size
2. Agitation
3. Heat
4. Viscosity
5. Vacuum

1.SPECIMEN SIZE
 Ideally tissue slice should be 3-4mm in
thickness
 Thicker specimens requires more time.

2. AGITATION
 The fluid exchange between processing fluid &
tissue is directly proportional to the surface area
of the tissue in contact with processing fluid.
 Agitation increases the flow of fresh solutions
around the tissue.
 In automated processor incorporates either
vertical or rotary oscillation or pressurized
removal & replacement of fluids

3. HEAT
 Heat increases the rate of penetration & fluid
exchange.
 Can cause shrinkage, hardening, brittleness of
the tissue.
 Temperature should be limited to 45degree
Celsius.

4.viscosity
 Low viscosity- has smaller molecules & a faster
penetration rate.
 High viscosity- larger molecules & slow
penetration.
 Solutions used in tissue processing,
dehydration, clearing have similar viscosities.
 Melted paraffin has a low viscosity.

5. Vacuum & PRESSURE
 Reduction of pressure increases the rate of infiltration.
• Less time is required
for embedding
• Embedding requires
care & supervision
• Removes air pockets in
porous tissue
• Too rapid evacuation of
air may cause rupture of
alveoli &
microscopically
mistaken for emphysema
• Clearing agents are
rapidly removed from
the tissue

 Tissue when exposed to heat or excessive
time in higher grade alcohols
Remove bound water
Shrinkage, parched earth effect, dry &
brittle tissue, abnormal staining.
 Incomplete dehydration impair the
penetration of clearing agents & non-
receptive to paraffin wax infiltration.

PRECAUTIONS:
 Wax used must contain no trace of clearing
agent.
 No dust particles.
 Immediately after embedding wax to be cooled
to decrease the wax crystal size.

MONITERING DEHYDRATION & CLEARING
 Monitor tissue dehydration & clearing by
analyzing TOF signals from specimens.
 In TOF, ultrasound(4MHz) acoustic pulses
transverse the tissue & are compared to pulses
through the reagent, the differential between
these wave forms are calculated as ΔTOF.

 TOF is used
 To monitor Neutral Buffered formalin diffusion
into tissue.
 To prevent over fixation / under fixation of
tissue & also dehydration & clearing

ROLE OF COPPER SULPHATE
 A layer of copper
sulphate ,1/4-1/2 inch
deep is placed at the
bottom of a
dehydrating beaker.
 Cover with 2-3 filter
papers to prevent
staining of the tissue

 Anhydrous copper sulphate removes water from
the alcohol.
 This speeds the process of dehydration of tissue
& prolong life of alcohol.
 The change of colour of copper sulphate from
white to blue indicate both alcohol & CuSo4 to
be changed

TISSUE PROCESSING
FOR SPECIAL STUDIES

TISSUE PROCESSING FOR IHC
 Fixation – 10% NBF
 Dehydration- graded alcohol
 Clearing- xylene
 Impregnation & embedding- low melting
point paraffin wax at 45 degree C.

TISSUE PROCESSING FOR ELECTRON MICROSCOPY
 Fixing:
 2-5% glutaraldehyde for 2-6 hours
 1% osmium tetraoxide for 60-90 minutes.
 Dehydration
 After fixation, tissue is washed to remove
excess osmium tetra oxide.
 Graded alcohol: 50%, 70%,95% & absolute
alcohol.

 Each change for 15min.
 Refrigerate overnight.
 Allow to return to room temperature.
 Before opening vial replace with fresh absolute
alcohol

TISSUE PROCESSING FOR ENZYME STUDIES
 Different processing is required for enzyme
studies
 Prevent them from getting damaged by routine
fixation.
 Difficulties:
• Oxidative enzymes in mitochondria are rapidly
damaged once blood supply is cut & hence
freezing is required.
• Freezing & thawing causes damage to lysosomal
enzymes hence controlled fixation is required.

CONCLUSION
 Max surface area 3×2cm.
 Not be more than 4mm thick (2-3mm).
 Fixative: 10% neutral buffer formalin.
 Dehydrating agent of choice :
1. Electron microscopy: Ethyl alcohol.
2. Microwave processing: Isopropyl alcohol.
3. Manual processing: Acetone.

 Clearing agent:
1. Xylene( more commonly used).
2. Chloroform : manual method.
 Impregnating agents:
1. Paraffin wax with ceresin.
 Embedding:
1. Most common: Paraffin embedding.
2. Rapid: Freeze drying

REFERENCE
 John D, Bancroft’s theory and practice of
histological techniques, 8th edition, 2019 73-83.
 C.F.A Culling, cellular pathology technique, 4th
ed,1985, 889-902.
 Nayak R, Histopathology techniques and its
management. 1st ed, new delhi; 2018, 41-68.

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