South Valley University

Name; Chama Kabaso

Student No. 201912010459
Module; CELLULAR PATHOLOGY
Assignment; 5
Question; Discuss the steps involved in tissue
processing stating the purpose of each stage and
the chemical used
Lecturer; Mr Kabasiya Musipili
Due Date; 05th May 2020
 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.
After the removal of a tissue sample from the patient, a series of physical and chemical
processes must take place 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. Every step in tissue processing is important;
from selection of the sample, determining the appropriate protocols and reagents to use, to
staining and final diagnosis.

STEPS OF TISSUE PROCESSING

• Fixation – stabilizes and hardens tissue with minimal distortion of cells.
• Dehydration – removal of water and fixative from the tissue.
• Clearing – removal of dehydrating solutions, making the tissue components receptive to the
infiltrating medium.
• Infiltrating – permeating the tissue with a support medium.
• Embedding – orienting the tissue sample in a support medium and allowing it to solidify.

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. Fixation must finish before subsequent steps in the
processing schedule are initiated. If fixation is not complete prior to processing, stations
should be designated on the processor for this purpose. If the tissue is inadequately fixed, the
subsequent dehydration solutions may complete the process, possibly altering the staining
characteristics of the tissue. 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. Care must be taken not to overfill the cassette, as this would impede the flow of
reagents around the tissue. If possible, larger and smaller pieces of tissue should be separated and
processed using different schedules. The most commonly used reagent for the fixation
of histological specimens is 10% neutral buffered formalin

CLEARING
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’.
The criteria for choosing a suitable clearing agent are:
• rapid penetration of tissues
• rapid removal of dehydrating agent
• ease of removal by melted paraffin wax
• minimal tissue damage
• low flammability
• low toxicity
• low cost
Most clearing agents are flammable liquids, which warrant caution in their use. The boiling point
of the clearing agent gives an indication of its speed of replacement by melted paraffin wax.
Fluids with a low boiling point are generally more readily replaced. Viscosity influences the
speed of penetration of the clearing agent. Prolonged exposure to most clearing agents causes the
tissue to becomebrittle. The time in the clearing agent should be closely monitored to ensure that
dense tissue blocks are sufficiently cleared and smaller more fragiletissue blocks are not
damaged. Cost should be considered, especially as it relates to disposal of thereagent. Since most
clearing agents are aromatichydrocarbons or short-chain aliphatichydrocarbons, environmental
issues must be addressed. Most institutions have a policy for the storage, disposal and safety
requirements for all flammables used in the laboratory.
CLEARING AGENTS SUITABLE FOR ROUTINE USE
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.
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.
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.

INFILTRATING
Paraffin wax
Paraffin wax continues to be the most popular infiltration and embedding medium in
histopathology laboratories. Paraffin wax is a mixture of longchained hydrocarbons produced in
the cracking of mineral oil. Its properties are varied depending on the melting point used, 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. 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 paraffinwax 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 protocol.

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. Most laboratories use modular
embedding centers, consisting of a paraffin dispenser, a cold plate, and a heated storage area for
molds and tissue cassettes. Paraffin wax is dispensed automatically from a nozzle into a suitably
sized mold. The tissue is oriented in the mold; a cassette is attached, producing a flat block face
with parallel sides. The mold is placed on a small cooling area to allow the paraffin. Wax to
solidify. The quick cooling of the wax ensures a small crystalline structure, producing fewer
artifacts when sectioning the tissue.
REFERENCES
1.Bancroft’s Theory and practice of Histological techniques 7 th Edition
2 Beltrami, C.A., Fabris, G., Marzola, A., et al., 1975. Staining of gastrin cells with lead
hematoxylin. Histochemical Journal 7, 95.

3.Carazzi, D., 1911. Eine neue Hämatoxylinlösung. Zeitschrift für wissenschaftliche

Mikroskopie und für mikrosko-pische Technik 28, 273.

4.Carson, F.L., 1997. Histotechnology: a selfinstructional text. American Society for Clinical
Pathology, Chicago, 6, 93

5.Cole, E.C., 1943. Studies in hematoxylin stains. Stain Technology 18, 125

6.Delafield, J., cited by Prudden, J.M., 1885. Zeitschrift für wissenschaftliche. Mikroskopie und
für mikroskopische Technik 2, 228.

7.Ehrlich, P., 1886. Fragekasten. Zeitschrift für wissenschaft-liche. Mikroskopie und für
mikroskopische Technik 3, 150.

8.Feldman, A., Dapson, R., 1985. Newsletter, Winter.ANATECH.

9.Feldman, A., Dapson, R., 1987. Newsletter, Winter.ANATECH