2. Tissue processing-Introduction:
• After the removal of a tissue sample from the patient, it is passed through a
series of physical and chemical processes to ensure that the final microscopic
slides produced are of a diagnostic quality.
• Tissues are exposed to a series of reagents that fix, dehydrate, clear, and
infiltrate the tissue.
• The tissue is finally embedded in a medium that provides support for microtomy.
• The quality of the structural preservation of tissue components is determined by
the choice of exposure times to the reagents during processing.
3. Principles of tissue processing
• Tissue processing is designed to remove all extractable water from the tissue,
replacing it with a support medium that provides sufficient rigidity to enable
sectioning of the tissue without parenchymal damage or distortion.
4. Factors influencing the rate of processing
• When tissue is immersed in fluid, an interchange occurs between the fluid
within the tissue and the surrounding fluid.
• The rate of fluid exchange is dependent upon the exposed surface of the tissue
that is in contact with the processing reagents.
• Several factors influence the rate at which the interchange occurs.
• agitation, heat, viscosity and vacuum.
5. Agitation
• Agitation increases the flow of fresh solutions around the tissue.
• Automated processors incorporate vertical or rotary oscillation, or pressurized
removal and replacement of fluids at timed intervals as the mechanism for
agitation.
• Efficient agitation may reduce the overall processing time by up to 30%.
6. Heat
• Heat increases the rate of penetration and fluid exchange.
• Heat must be used sparingly to reduce the possibility of shrinkage,
hardening or embrittlement of the tissue sample.
• Temperatures limited to 45°C can be used, but higher temperatures
may be deleterious to subsequent immunohistochemistry.
• Viscosity
• The smaller size of the molecules in the solution, the faster the rate of
fluid penetration (low viscosity).
• Conversely, if the molecule size is larger, the rate of exchange is
slower (high viscosity).
7. Vacuum
• Using pressure to increase the rate of infiltration decreases the time
necessary to complete each step in the processing of tissue samples.
• Vacuum will remove reagents from the tissue, but only if they are more
volatile than the reagent being replaced.
• Vacuum used on the automated processor should not exceed 50.79 kPa to
prevent damage and deterioration to the tissue.
• Vacuum can also aid in the removal of trapped air in porous tissue.
• Impregnation time for dense, fatty tissue can be greatly reduced with the
addition of vacuum during processing.
8. Stages of tissue processing
1. Fixation – stabilizes and hardens tissue with minimal distortion of cells.
2. Dehydration – removal of water and fixative from the tissue.
3. Clearing – removal of dehydrating solutions, making the tissue
components receptive to the infiltrating medium.
4. Infiltrating – permeating the tissue with a support medium.
5. Embedding – orienting the tissue sample in a support medium and
allowing it to solidify.
9. 1. Fixation
• Preserving cells and tissue components with minimal distortion is the most
important aim of processing tissue samples.
• Fixation stabilizes proteins, rendering the cell and its components resistant to
further autolysis by inactivating lysosomal enzymes.
• It also changes the tissues’ receptiveness to further processing.
• The size and type of specimen in the tissue cassette determines the time needed
for complete fixation and processing.
• The tissue should be dissected to 2–4 mm in thickness.
10. Dehydration
• Dehydration is the removal of ‘free’ unbound water and aqueous fixatives from the
tissue components.
• Many dehydrating reagents are hydrophilic that interact with the water molecules in the
tissue by hydrogen bonding.
• Other reagents affect dehydration by repeated dilution of the aqueous tissue fluids.
• Dehydration should be accomplished slowly.
• If the concentration gradient is excessive, diffusion currents across the cell membranes
may increase the possibility of cell distortion.
11. Dehydration
• For this reason, specimens are processed through graded strengths of reagents
of increasing concentration.
• Excessive dehydration may cause the tissue to become hard, brittle and
shrunken.
• Incomplete dehydration will impair the penetration of the clearing reagents into
the tissue, leaving the specimen soft and non-receptive to infiltration.
12. Dehydrating fluids
1. Ethanol (C2H5OH): Ethanol is a clear, colorless, flammable liquid.
• It is hydrophilic, miscible with water and other organic solvents, fast-acting and
reliable.
• Aside from its human health-risk potential, ethanol is taxable, controlled by
many governments, and therefore requires careful record keeping.
• Graded concentrations of ethanol are used for dehydration; the tissue is
immersed in 70% ethanol in water, followed by 95% and 100% solutions.
13. Dehydrating fluids
2. Industrial methylated spirit (denatured alcohol)
• This fluid has the same physical property as ethanol.
• Denatured alcohol consists of ethanol, with the addition of methanol (about
1%), isopropyl alcohol or a combination of alcohols.
• For purpose of tissue processing it is used in the same manner as ethanol.
3.Methanol (CH3OH):
• Methanol is a clear, colorless and flammable fluid that is miscible with water,
ethanol and most organic solvents.
• It is highly toxic but can be substituted for ethanol in processing protocols.
14. Dehydrating fluids
4. Propanol, isopropyl alcohol (CH3CHOHCH3):
• Isopropyl alcohol is miscible with water, ethanol and most organic solvents.
• Isopropyl alcohol does not cause over-hardening or shrinkage of the tissue.
5. Acetone (CH3COCH3):
• Acetone is a clear, colorless, flammable fluid that is miscible with water, ethanol
and most organic solvents.
• It is rapid in action, but has poor penetration and causes brittleness in tissues if
its use is prolonged.
• Acetone removes lipids from tissue during processing.
15. Additives to dehydrating agents
• When added to dehydrating agents, phenol acts as a softening agent for hard
tissues such as tendon, nail, and dense fibrous tissue and keratin masses.
• Phenol (4%) should be added to each of the 95% ethanol stations.
• Alternatively, hard tissue can be immersed in a glycerol/alcohol mixture.
16. Clearing
• Clearing means appearance of tissue after it has been treated by the clearants to
remove the dehydrating agent.
• Clearing reagents act as an intermediary between the dehydration and infiltration
solutions.
• They should be miscible with both solutions.
• Most clearants are hydrocarbons with refractive indices 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’.
17. Clearing
• The criteria for choosing a suitable clearing agent are:
1. rapid penetration of tissues
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
18. Clearing agents for routine use
1. Xylene: Xylene is a flammable, colorless liquid with a characteristic petroleum or
aromatic odor, which is miscible with most organic solvents and paraffin wax.
• It is suitable for clearing blocks that are less than 5 mm in thickness and rapidly
replaces alcohol from the tissue.
• Overexposure to xylene during processing can cause hardening of tissues.
• It is most commonly used in routine histology laboratories and is also recyclable.
19. Clearing agents for routine use
2. Toluene: This has similar properties to xylene, although it is less damaging with
prolonged immersion of tissue.
• It is more flammable and volatile than xylene.
3. Chloroform: Chloroform is slower in action than xylene but causes less
brittleness.
• Thicker tissue blocks can be processed, greater than 1 mm in thickness.
• Tissues placed in chloroform do not become translucent.
• It is non-flammable but highly toxic, and produces highly toxic phosgene gas
when heated.
• It is most commonly used when processing specimens of the central nervous
system.
20. Impregnation or infiltration
• After clearing tissue biopsies are transferred to some solidifying medium which
replaces the clearing agent and then completely fills the natural cavities, spaces
and interstices of the tissues.
• An impregnating media must enter the tissue spaces in liquid form and then
solidify to support cells.
21. Impregnating media
• Paraffin wax:
• Paraffin wax is the most popular infiltration and embedding medium in
histopathology laboratories.
• Paraffin wax is a mixture of long-chained hydrocarbons produced in the cracking
of mineral oil.
• Its properties are varied depending on the melting point ranging from 47 to 64°C.
• Paraffin wax permeates the tissue in liquid form and solidifies rapidly when
cooled.
• The tissue is impregnated with the medium, forming a matrix and preventing
distortion of the tissue structure during microtomy.
• It has a wide range of melting points, which is important for use in the different
climatic regions of the world.
22. Impregnating media
• To promote desirable ribboning during microtomy, paraffin wax of suitable
hardness at room temperature should be chosen.
• Heating the paraffin wax to a high temperature alters the properties of the wax.
• Higher melting point paraffin wax provides better support for harder tissues, e.g.
bone, can allow production of thinner sections, but may cause difficulty with
ribboning.
• Lower melting point paraffin wax is softer and provides less support for harder
tissues.
• It is more difficult to obtain thinner sections but ribboning is easier.
• Paraffin wax is inexpensive, provides quality sections and is easily adaptable to a
variety of uses.
• Paraffin wax is compatible with most routine and special stains, as well as
immunohistochemistry protocols.
23. Paraffin wax additives
• Paraffin waxes that contain plasticizers or other resin additives are commercially
available.
• These additives create paraffin waxes with selectable hardness compatible with
the tissue to be embedded.
• Substances added to paraffin wax in the past include beeswax, rubber, ceresin,
plastic polymers and diethylene glycol distearate.
• Many of these additives had a higher melting point than paraffin wax,
consequently making the tissue more brittle.
24. Alternative embedding media
• Resin:
• Resin is used exclusively as the embedding medium for electron microscopy,
ultra-thin sectioning for high resolution and also for undecalcified bone.
• Celloidin:
• The use of celloidin or LVN (low viscosity nitro-cellulose) is discouraged because
of the special requirements needed to house the processing reagents.
• It is rarely used.
25. Paraffin wax embedding
• Embedding involves the 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 while
facilitating sectioning.
26. Paraffin wax embedding
• Orientation of tissues:
• Specimen orientation during embedding is important for the demonstration of
proper morphology.
• Incorrect orientation may result in diagnostic tissue elements being damaged
during microscopy or not being evident for pathology review.
• Orientation of the tissue should offer the least resistance of the tissue against the
knife during sectioning.
• A margin of embedding medium around the tissue assures support of the tissue.
27. Paraffin wax embedding
• Tubular structures: cross section of the wall and lumen should be visible;
arteries, veins, fallopian tube and vas deferens samples.
• Skin biopsies: shave punch or excisions, cross section of the epidermis, dermis
and subcutaneous layers must be visible.
• Intestine, gallbladder, and other epithelial biopsies: cut in a plane at right angles
to the surface, and oriented so the epithelial surface is cut last, minimizing
compression and distortion of the epithelial layer.
• Muscle biopsies: sections containing both transverse and longitudinal planes.
• Multiple pieces of a tissue are oriented side by side with the epithelial surface
facing in the same direction.
