TISSUE PROCESSING

INTRODUCTION

• Steps required to take an

animal or human tissue
from fixation to the state
where it is completely
infiltrated with a suitable
histological wax and can be
embedded ready for
section cutting on the
microtome
 Manual

Automatic
PRINCIPLE

Provides sufficient
Remove all Replacing it with rigidity to enable
extractable a support sectioning of the
water from the tissue without
medium parenchymal
tissue damage or distortion
 • To equalise concentrations inside and
outside the blocks of tissue depends on
Fick’s law
• The rate of solution diffusion through the
FICK’S LAW tissues is proportional to the concentration
gradient (between the concentrations of
the fluids inside and outside of the tissue)
as a multiple of temperature dependant
constants for specific substances
FACTORS INFLUENCING RATE OF PROCESSING

AGITATION HEAT VISCOSITY VACUUM

AGITATION

INCREASES THE FLOW OF FRESH AUTOMATED PROCESSORS EFFICIENT AGITATION REDUCES

SOLUTIONS AROUND THE TISSUE INCORPORATE VERTICAL OR ROTARY OVERALL PROCESSING TIME BY 30%
OSCILLATION AT TIMED INTERVALS
HEAT

It has to be used
Increases rate of
sparingly to reduce
penetration and fluid
shrinkage, hardening or
exchange
embrittlement

45o C is the most

acceptable temperature
VISCOSITY

Smaller sized molecules will penetrate faster as

compared to larger molecules

Rate of exchange is higher

Paraffin has lower viscosity – enhances the rapidity

of impregnation
VACUUM

Vacuum will remove

reagents from tissue
Pressure can be used to
only if they are more
increase rate of filtration
volatile than the reagent
being replaced

It also aids in removal of

trapped air in porous
tissue
STAGES

FIXATION DEHYDRATION CLEARING INFILTRATING EMBEDDING

 • Stabilises and
hardens tissue with
minimal distortion of
cells
• Preservation of cell
architecture
• Prevent autolysis
FIXATION and putrefaction
• Thickness of tissue
should be 2-4mm
• Most commonly
used fixative agent –
10% NBF
FIXATIVE
• Is defined as a substance which prevents
post mortem changes and preserves the
morphological and chemical characteristics
of the cell
• The mechanism and principles by which
specific fixatives act fall into several broad
groups- Cross linking, dehydration, effect of
acids, salt formation and heat
DEHYDRATION
Dehydrating agents are
Removal of free
hydrophilic and they
unbound water and
First step in processing interact with water
aqueous fixatives from
molecules in the tissue
tissue components
by hydrogen bonds

Agents used- Ethanol,

Excessive dehydration
Gradual process with ethanol acetone,
may cause tissue to
increasing amount of methanol, isopropyl,
shrink, become hard
alcohol concentrations glycol, denatured
and brittle
alcohols
ETHANOL

• Clear, colourless flammable liquid

• Hydrophilic, miscible with water and
other organic solvents, fast acting,
reliable
• Graded concentrations are used
• First- 70% ethanol in water, 95% and
100%
• Ensures total dehydration
• Reagent of choice for Electron
Microscopy
• Delicate tissues to be processed in 30%
ethanol
 • Clearing reagents act as intermediaries
between dehydration and infiltration
• They should be miscible with both solutions
• They are usually hydrocarbons with
CLEARING refractive indices similar to protein
• Once they completely replace all the
alcohol in the tissues, it gives tissue a
translucent appearance
 Rapid penetration of tissues

Rapid removal of dehydrating agent

CRITERIA FOR Ease of removal by molten paraffin wax
SELECTION OF Minimal tissue damage
APPROPRIATE Low flammability
AGENT Low toxicity

Low cost
 Usually flammable liquids

Prolonged exposure causes tissue to become brittle

CLEARING Time should be carefully monitored

AGENTS Usually aromatic hydrocarbons, short chain aliphatic -

environmental toxicity

Common agents- Xylene, toluene, chloroform, xylene

substitutes limonene reagents

Volume should be 50-100 times the volume of the

specimen
XYLENE
• Flammable, colourless liquid with
characteristic aromatic odour
• Miscible with most organic solvents
and paraffin wax
• Suitable for clearing blocks which are
less than 5mm thickness
• Rapidly replaces alcohol from tissue
• Overexposure can cause hardening of
tissues
• Most commonly used clearing agent
• Recyclable
TOLUENE
• Similar properties to xylene
• Less damaging when used for longer
time periods
• More flammable and volatile than
xylene
CHLOROFORM
• Slower in action than xylene
• Causes less brittleness
• Thicker tissues blocks > 1mm can be
processed
• Tissues do not become translucent
• Non flammable, highly toxic-
phosgene gas when heated
• Most commonly used for Central
Nervous System specimens
 Aliphatic hydrocarbons

XYLENE Differ in the number of carbon atoms

SUBSTITUTES Short chain aliphatic have the same evaporation

properties as that of xylene, no affinity for water

Long chain aliphatic do not evaporate rapidly and

may cause contamination of the paraffin wax
CEDARWOOD OIL

• Best reagent for delicate tissues

• Least hardening effect
• Can be used for months without
tissue damage
INFILTRATING
• Process by which empty space in tissues and cells are
filled with wax after clearing
• Usually done with paraffin wax at temperatures between
47o C to 64o C
• Paraffin wax permeates the tissue in liquid form and then
solidifies rapidly when cooled
• To promote desirable ribboning of sections, paraffin wax
of suitable hardness at room temperature must be chosen
• Higher melting point waxes provide greater support for
harder tissues – bone
• Lower melting point waxes are used for softer specimens
• Paraffin wax is compatible with most routine and special
stains as well as immunohistochemistry protocols
 Resin Agar
ALTERNATIVE
EMBEDDING
MEDIA
Gelatin Celloidin
 Used exclusively as
embedding media for
electron microscopy
RESIN
Suitable for ultra thin
sectioning for high
resolution and also for
undecalcified bone
 Agar gel alone is never used, it’s
main use is as a cohesive agent for
small friable tissue pieces post
fixation- double embedding

Fragments are embedded in molten

AGAR agar and allowed to solidify for
sectioning

Special method- Millipore

technique
GELATIN

Primarily used in
production of sections of
whole organs using Gough- Rarely used
Wentworth technique in
frozen sectioning
CELLOIDIN
Also called LVN- Low
Viscosity Nitrocellulose

Needs special requirement

of processing reagents

Limited to use in
neuropathology

Rarely used
ORIENTATION OF TISSUE
• Specimen orientation during embedding is most
important for demonstration of proper morphology
• Incorrect orientation may result in diagnostic tissue
elements being damaged during microscopy
• Orientation of tissue should offer least resistance
against knife during sectioning
• Certain tissues require special orientation-
• Tubular structures, Skin biopsies, GIT biopsies, muscle
biopsies
EMBEDDING

• Involves enclosing of properly

processed, correctly oriented
specimens in a support medium
that provides external support
during microtomy
• The embedding media must fill
the matrix within the tissue,
supporting cellular components
• The medium should provide
elasticity, resisting section
distortion during sectioning
 • Most popular embedding medium in
histopathology

PARAFFIN • It is a mixture of long chain hydrocarbons

produced in cracking of mineral oil
• Certain additives are added to increase its
WAX hardness
• Substances include- beeswax, rubber, ceresin,
plastic polymers and diethylene glycol Di stearate
PROPERTIES
• Soluble in processing fluids
• Suitable for sectioning and ribboning
• Molten between 30o C and 60o C
• Translucent/ transparent
• Stable
• Homogenous
• Capable of flattening after ribboning
• Non toxic, odourless
• Easy to handle
• Inexpensive
• Most laboratories use a
modular embedding centers
consisting of –
• Paraffin dispenser
• Cold plate
• Heated storage area for
molds and tissue cassettes
TYPES OF
MOLDS
• Leuckhart’s L pieces
• Glass petri dishes
• Metal Petri dishes
• Paper Boats
FAULTS REASON REMEDY

Brittle or Hard tissue Prolonged Fixation Tissue to be softened by soaking in a small dish or
Prolonged dehydration bowl containing water with detergent, phenol or
Prolonged clearing Molliflex
Prolonged paraffin infiltration in overheated
paraffin oven
Drying of tissues before fixation

Clearing agent turns milky Incomplete dehydration Repeat dehydration with absolute alcohol and clear
again

On trimming, tissue smells of clearing agent Incomplete clearing due to insufficient Re- impregnation
impregnation

Opaque tissue Insufficient clearing Repeat clearing

Tissue shrinkage during trimming Insufficient dehydration Repeat whole procedure

Soft tissue Incomplete fixation Repeat fixation

Crystalline wax Contaminated wax Re-embed in freshly filtered wax

Paraffin block after cooling becomes moist and Insufficient paraffin impregnation Repeat impregnation and then re-embed
crumbles
DECALCIFICATION
 • Technique for removing minerals from bone or
other calcified tissues so that good quality
paraffin sections can be prepared that will
preserve all the essential microscopic elements
• Carried out after the tissue has been thoroughly
INTRODUCTION fixed, prior to routine tissue processing
• Decalcified sections are used for the
examination of bone marrow and for diagnosis
of tumours, infections, etc
 • Buffered Formalin
FIXATIVES FOR • Zinc Formalin
BONE • B5 fixative
• Formal acetic alcohol (Davidson’s Fixative)
SAMPLES • Bouin’s solution
 • 3 main categories are present-
DECALCIFYING 1. Agents based on strong mineral acid
AGENTS 2. Agents based on weak organic acids
3. Chelating agents
STRONG ACIDS
• 10% Hydrochloric or nitric acid are the
most rapid in action, however if used for
an excessive time can lead to loss of
nuclear staining and maceration of tissues
DECALCIFIER FORMULA PROPERTIES
Nitric Acid 5% in distilled water Rapid in action, exceeding end point will impair
staining

Perenyi’s Fluid (1882) 10% nitric Acid 40ml A traditional decalcifier that decalcifies slowly than
0.5% chromic acid 30 ml aqueous nitric acid
Absolute Alcohol 30ml Quite rapid in action and exceeding end point will
impair staining

Hydrochloric Acid 5% - 10% in distilled water Formalin should be washed from specimen before
placing in HCl to avoid formation of bischloromethyl
ether( a carcinogen)
Rapid in action, exceeding end point will impair
staining

Von ebner’s solution Sodium chloride saturated solution 10 ml Rapid in action, exceeding end point will impair
Distilled water 42ml staining
Hydrochloric Acid 8ml
WEAK ACIDS
• Formic acid is popular and widely used for decalcification
• Can be used as simple 10% aqueous solution or combined with
formalin/ buffer
• Time of action is slower, it is much gentle and is less likely to
interfere with nuclear staining
• Trichloroacetic acid (TCA), Picric acids
DECALCIFIER FORMULA PROPERTIES

Formic Acid 10% in distilled water A simple effective decalcifier

Evans and Krajian Formic Acid 25ml An effective formic acid decalcifier buffered with
Sodium Citrate 10g citrate
Distilled water 75ml

Kristensen Formic acid 18ml An effective formic acid decalcifier buffered with
Sodium formate 3.5g formate
Distilled water 82ml

Gooding and Stewart Formic acid 25 ml A formic acid decalcifier with added formalin,
40% Formaldehyde 5ml claimed to fix and decalcify
Distilled water 75ml
 • Chelating agents work by capturing the calcium ions from
the surface of the apatite crystal, slowly reducing its size
• Process is very slow but very gentle (decalcification may
take weeks to months)

CHELATING • Not suitable for urgent specimens

• Suitable for research applications where high quality
AGENTS morphology is required for particular molecular elements
• Applications- IHC, FISH, PCR
• Used in a concentration of 14% as a mineralised solution
• Rate of decalcification is pH dependant. Optimum pH – 7;
Rapid action- 10
DECALCIFIER FORMULA PROPERTIES

Neutral EDTA EDTA disodium salt 250g Acts slowly but causes
Distilled water 1750 ml little tissue damage
Conventional stains are
Bring to pH 7 by adding largely unaffected
sodium hydroxide 25g
FACTORS AFFECTING RATE OF
DECALCIFICATION

Concentration
Temperature
Agitation
Fluid Excess
 Sonication used with EDTA – accelerate
decalcification, temperature has to be carefully
controlled
TECHNIQUES FOR
IMPROVING THE Microwave used with HCl decalcifiers- raised
temperature may damage morphology and cause
EFFICACY OF staining artifacts
DECALCIFYING
AGENTS Addition of ion- exchange resins- take up the ionised
calcium maintaining the effectiveness of the acid

Electrolytic decalcification – acid decalcifiers

DETERMINATION OF END POINT
• X ray of specimen
• Chemical test- Ammonium Oxalate solution
• Physical tests- manipulation, bending, probing, trimming of
specimen to feel for remaining calcified areas
• Weighing of specimen after rinsing and blotting – effective
for large specimens
• Slow decalcification- removed from decalcifier, rinsed
thoroughly and placed back into formalin; Store in
refrigerator at 4o C
• Post decalcification specimen to be washed thoroughly in
tap water, application of alkaline solutions
 It is a method of dealing with small unexpected
deposits of calcium that maybe encountered in
paraffin blocks
Once calcification is exposed, the tissue block is
removed from the microtome and placed face
SURAFCE down in an acid decalcifier for 15-60 mins
DECALCIFICATION This will allow the decalcifier to penetrate a
small distance into the block and dissolve the
calcium
The block is then thoroughly rinsed in water to
remove residual acid
Ground Sections
• What can you not see in a decalcified specimen?
Preparing ground sections
• Lathe abrasion

• Hard tissue microtome

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