30-04-2014, 12:26 PM
Histopathology Techniques: Tissue Processing and Staining
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Incisional biopsy:
In this method only a portion or wedge of tissue from a large lesion is taken and therefore, the
procedure is strictly a diagnostic nature. It is performed when removal of entire lesion is
impossible and often performed prior to major surgical procedure.
Excisional Biopsy:
In this technique, the entire lesion is removed, usually with a rim of normal tissue and therefore,
the procedure serves the diagnostic and therapeutic function. Excising the entire lesion ensures
sufficient tissue for histopathological examination, lessen the risk of tumour dissemination and
eliminate sampling errors. It is performed when the lesion is smaller in size.
Punch Biopsy:
It is done by biopsy forceps. It is performed in the lesion of uterine cervix, oral cavity,
esophagus, stomach, intestine and bronchus.
Some General Rules for the biopsy Procedure:
1. The larger the lesion, the numerous the biopsies that should be taken from it because of
the variability in the pattern that may exists and the fact that the diagnostic areas may be
present only focally.
2. In ulcerated tumour, biopsies of the central ulcerated areas may show only necrosis and
inflammation. Biopsies should be taken from the periphery that includes normal and
diseased tissue.
3. The biopsies should be deep enough that the relationship between tumour and stroma can
be properly assessed.
4. Deeply seated lesions are sometimes accompanied by a prominent peripheral tissue
reaction which may be characterized by chronic inflammation, hyperemia, fibrosis,
calcification and metastatic bone formation. If the biopsy is too peripheral, this may be
the only tissue obtained.
5. When several fragments of tissue are obtained they should be sent to the pathology
laboratory and all of them submitted for microscopic examination.
6. Crushing or squeezing of the tissue with forceps should be carefully avoided.
7. Once the biopsy is obtained, it should be placed immediately into container with adequate
volume of fixative.
Handling of Specimen
Specimen should be transported in glass, plastic or metal container or in a plastic bag in 10%
formalin. If formalin is not available at hand, place the specimen in refrigerator at 4oC to slow
down autolysis. The container should have an opening larger enough so that the tissue can be
removed easily after it has hardened by fixation. However, fresh material is needed for the
following purpose:
1. Frozen section
2. Immunocytochemistry
3. Cytological examination
4. Microbiological sampling before histopathology
5. Chromosome analysis
6. Research purpose
7. Museum display
Tissue Processing:
In order to cut thin sections of the tissues, it should have suitable hardness and consistency when
presented to the knife edge. These properties can be imparted by infiltrating and surrounding the
tissue with paraffin wax, colloidin or low viscosity nitrocellulose, various types of resins or by
freezing. This process is called tissue processing. It is done in stages. It can be subdivided into
dehydration, clearing, impregnating and embedding. It is important that all specimens are
properly labeled before processing is started.
For labeling, pen containing ordinary ink should not be used. Printed, graphite pencil written,
type-written or India ink written labels are satisfactory.
A system of transportation is required to carry the tissue through various steps in processing. The
cut specimens are put in muslin cloth together with their labels and are then transported from
reagent to reagent in metal containers that have perforated walls, so that the reagent enters into
the tissues.