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Pathology Unit's laboratory receives several different samples from ophthalmic surgeries:
- Connective tissue
Apart from these types of biopsies, it's also possible to send the lab different kinds of cytologies (mainly touch imprint and vitreous cytologies) to complement clinical diagnoses. Biopsy samples undergo a formaldehyde fixation process, treatment with alcohols and xylenes and paraffin inclusion to be able to obtain 3-to-4 micron microtome excisions. Following this, they are dyed with haematoxylin and eosin, ready to be examined under a microscope and obtain a diagnosis.
There are, basically, three types of complementary techniques:
- Hybridization in situ
Histochemical techniques enable us to detect special tissue components and substances through chemical reaction of certain reagents with the tissue elements we attempt to demonstrate. The more common ones are Periodic Acid-Schiff Stain for glycogen and neutral mucopolysaccharides, Alcian Blue for acid mucopolysaccharides, Prussian Blue for iron, Congo Red for amyloid, Zielh-Neelesen for microbacteries; Trichrome for muscle tissue and collagen.
Immunohistochemical techniques are based on the Antigen-Antibody reaction. Tissues are incubated with specific antibodies against the concrete proteins whose presence we aim to demonstrate. These antibodies are marked with a chromogen in such a way that, in the presence of antigens, they form complexes visible under the microscope. There exists a broad variety of antibodies available. By way of example, they are used against intermediate filaments of the cytoskeleton as keratins, desmin, vimentin; melanocytic markers (S100, HMB54, Mart1), cell-proliferation markers (ki67), lymphoid line markers (CD20, CD3, CD30), etc.
In situ hybridization techniques are based on the DNA and RNA chains's abiity to join other chains with complementary sequences. One uses probes with a known sequence of nucleotides in order to link them to cell DNA or RNA when complementary sequences exist. Such probes are marked with a substance (usually fluorescent) that is visible under the microscope, which explains why we talk about in situ hybridization techniques with fluorescence or FISH. They are used, for instance, to find cells with chromosomal gains or losses, or rearrangements of parts between them, which helps detect certain tumours.