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Histopathology Laboratory

Tissue fixation and processing
Paraffin block sectioning
Frozen block sectioning
Tissue staining

Gross Examination

Once a specimen is received in the Histopathology Laboratory, it is identified by its own unique accession number. The gross examination is important because once the specimen has been sectioned, all gross information is lost. At this time the gross specimen is weighed, measured, and may be photographed or X-rayed. If necessary, the margins are inked to determine the adequacy of surgical margins. Finally, a report of the macroscopic findings are dictated

Tissue that has been cut and placed in cassette

Dissection

Following protocols for the dissection of various organs, the pathologist or pathologist's assistant determines which areas to sample for microscopic examination. 3mm sections of tissue are cut from the specimen and placed into cassettes, then returned to formalin

Fixation

Specimens are fixed in 10% buffered formalin for several hours to denature the proteins and harden them. This prevents further decomposition of the tissue by arresting cell ****bolism. Some specimens are fixed overnight, prior to being examined grossly, to make them easier to handle and section

Dehydration

Tissue processor for dehydration

Tissues must be dehydrated before they can be infused with paraffin. In the automated tissue processor, sectioned tissue is submerged in a series of alcohol baths to remove water. The alcohol must then be cleared with a clearing agent, such as toluol, which is miscible with paraffin. This process is performed overnight so that the tissue is ready to be embedded with paraffin the next morning

Tissue embedded in molten paraffin

Paraffin Embedding

The tissue is placed in molten paraffin at 52 - 56°C for several minutes so that once the paraffin cools, the tissue and block will be hard enough to cut. Depending on the tissue, it will need to be embedded in the paraffin in a specific orientation. For example, a tubular structure is positioned so that when it is cross-sectioned, the entire lumen is visible

Cutting

The paraffin blocks containing the tissue are cut producing 5µm sections with a microtome. This piece of equipment has a set mechanism for advancing the block across a very sharp knife. The cut sections are floated on a water bath to remove wrinkles, then transferred by hand to glass slides

Cutting sections with the microtome

Sections in waterbath picked up on slides

De-Paraffinization

In order for the water-soluble dyes to penetrate the tissue, the sections must be rehydrated. Toluol is used to remove the paraffin, then a series of alcohol washings rehydrates the tissue

Staining

The slides are stained to differentiate the nuclear material and connective tissue from the rest of the tissue. The KGH histopathology laboratory uses the hematoxylin-phloxine-saffron (HPS) stain which is more effective than the regular hematoxylin-eosin (H & E) stain used by most laboratories. HPS stains the nuclei blue; the cytoplasm, muscle, and myelin red; and the connective tissue yellow

Slides of surgical specimens

Mounting

The tissue is again dehydrated with alcohol and toluol to preserve it indefinitely. The glass coverslip is applied by an automated machine. The slides are now ready to be viewed by the pathologist.

De-Calcification

When specimens contain calcium, such as samples of bone or bone marrow, they must be de-calcified prior to cutting. Once the specimen is dissected into small sections and placed in plastic cassettes, it is de-calcified in RDO for approximately 4 hours

Special Stains

Sometimes the pathologist will order a special stain which highlights various features of the tissue or chemicals present

Examples of special stains are

Chemicals for special stains

Carbohydrate Stains
ex: PAS (periodic acid schiff) stain

Pigment Stains
ex: Prussian blue stain for iron

Micro-organism Stains
ex: Giemsa stain for H.pylori bacterium

Connective Tissue Stains
ex: Gordon and Sweet stain for reticulin

Pathology resident examing a frozen section

Frozen Sections

The frozen section laboratory is located close to the operating room, so that tissue removed in surgery can be processed immediately. When the specimen is received, it is labelled with its own unique accession number. The specimen is examined grossly and a section of tissue is sampled. The tissue sample is frozen in a Histobath containing isopentane at a temperature of -50°C. It is then transferred to a cryostat; a refrigerated box containing a microtome which cuts the tissue into 5µm sections. These sections are then mounted on slides and stained to differentiate the nuclear material from the rest of the tissue. The section is fixed in acidified alcohol, stained with hematoxylin and eosin, dehydrated in ethanol, and cleared in toluol. The entire process takes approximately 10 minutes to complete, allowing the pathologist to microscopically examine the specimen and quickly provide a diagnosis to the surgeon

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