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Lab 3: Section Cutting and Frozen Section. Objectives:. 1. To ensure good section cutting and frozen section. 2. To troubleshoot problems during section cutting and frozen section. 3. To familiarize the staff with the equipment used for section cutting and frozen section. Overview:.
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Objectives: 1. To ensure good section cutting and frozen section. 2. To troubleshoot problems during section cutting and frozen section. 3. To familiarize the staff with the equipment used for section cutting and frozen section.
Overview: • Tissues are sectioned using a microtome. • Turn on the water bath and check that the temp is 35-37ºC. • Use fresh deionized water (DEPC treated water must be used if in situ hybridization will be performed on the sections). • Blocks to be sectioned.
Cont. • Place a fresh blade on the microtome; blades may be used to section up to 10 blocks, but replace if sectioning becomes problematic. • Insert the block into the microtome chuck so the wax block faces the blade and is aligned in the vertical plane.
Face the block by cutting it down to the desired tissue plane and discard the paraffin ribbon. • If the block is ribboning well then cut another four sections and pick them up with forceps or a fine paint brush and float them on the surface of the 37ºC water bath. • Float the sections onto the surface of clean glass slides.
Cont… • If the block is not ribboning well then place it back on the ice block to cool off and firm up the wax. • If the specimens turn into fragments when placed on the water bath then it may be too hot. • Place the slides with paraffin sections on the warming block in a 65°C oven for 20 minutes (so the wax just starts to melt) to bond the tissue to the glass. • Slides can be stored overnight at room temperature.
Cont… • Most sectioning in routine histopathology departments is done with a microtome producing sections of ~3μm thickness, from tissue that has been embedded in wax. • A number of devices are available for cutting sections.
Microtomes • These are mechanical devices for cutting uniform sections of tissue of appropriate thickness. • Ultramicrotome>>>> 50-100nm
Types of microtome 1. Hand microtomes 2. Rocking microtome. 3. Rotary microtome. 4. Freezing microtome.
Cont. 5. Base sledge microtome. 6. Vibrating knife microtome. 7. Sliding microtome.
Rotary microtome • Designed for cutting celloidin embedded tissue blocks. • The Knife or blade is stationary, specimen slides under it during sectioning. • Also used for paraffin-wax embedded sections.
Section adhesives • An adhesive is a substance which can be smeared on to the slides so that the sections stick well to the slides. • Most of the tissue sections which are adequately thin and thoroughly dried without any air bubble trapped under them do not require an adhesive, as in case of routine H and E staining.
Cont… • But for histochemical methods requiring alkaline solutions, e.g. ammonia tend to remove sections from slide for such cases adhesive is required. • Also adhesive is required for tissues like brain, spinal cord, blood clot, decalcified tissues which have a tendency to detach themselves from the slide. • Tissue impregnated with ester wax also requires section adhesive.
Common Problems with Sectioning Cutting Problems: • Cut on an angle: Angled cuts can be identified in the following ways: • The section or cells within it are oval in shape. • One can focus through several cell layers in one area of the section. • Part of the section appears to be “smeared”.
Cont… 2. Cut too thin: This problem can be identified if a section has a part missing, and/or it is not completely round. 3. Cut too thick: This problem can be identified if boundaries within the section appear exceptionally thick, or if you are able to focus through severalcell layers across the whole section.
Cont… 4. Sections cut in half: The problem here is that there are too many sections in the drop of water, and convection is bringing them back towards the razor where they are being cut in half.
Frozen Section • A thin slice of tissue that is cut from a frozen specimen and is often used for rapid microscopic diagnosis section and a histologic section of tissue that has been frozen by exposure to dry ice. • The frozen section procedure is a pathological laboratory procedure to perform rapid microscopic analysis of a specimen. It is used most often in oncological surgeries.
Cont… • The technical name for this procedure is cryo-sectioning. • The quality of the slides produced by frozen section is of lower quality than formalin fixed, wax embedded tissue processing. • While diagnosis can be rendered in many cases, fixed tissue processing is preferred in many conditions for more accurate diagnosis.
Uses from frozen section: The principal use of the frozen section procedure is the examination of tissue while surgery is taking place. • In the performance of Mohs surgery - a simple method for 100% margin control of a surgical specimen.
If a tumor appears to have metastasized, a sample of the suspected metastasis is sent for cryo-sectioning to confirm its identity. If the tumor has metastasized, surgery is usually not curative, and the surgeon will choose a more conservative surgery, or no resection at all.
Cont… • If a tumor has been resected but it is unclear whether the surgical margin is free of tumor, an intraoperative consultation is requested to assess the need to make a further resection for clear margins. • In a sentinel node procedure, a sentinel node containing tumor tissue prompts a further lymph node dissection, while a benign node will avoid such a procedure. • If surgery is explorative, rapid examination of a lesion might help identify the possible cause of a patient's symptoms.
Cont… • Rarely, cryo sections are used to detect the presence of substances lost in the traditional histology technique, for example lipids. They can also be used to detect some antigens masked by formalin.
Embedding the tissue: The selected piece of tissue is then placed on a metallic holder and must be oriented a certain way so that the future section will reveal proper spatial relationships, this orientation depends on the question being asked. Sometimes orientation is not important; at other times it is of paramount importance. The tissue is embedded in OCT mounting medium and is then placed either in cooled 2-methyl butane or the cryostat machine where it is properly frozen.
Cryostat:The machine, which cuts the tissue, is the cryostat. Certain things should be routinely checked in the operation of this machine: a) Temperature: The temperature should be at -20°C for most tissues. For tissues with a large fat component, -40°C is optimal. This temperature is critical for optimal sectioning.
Cont… • Too high, i.e., -10°F and the tissue will not stay frozen and firm and will not cut properly. • Too cold, i.e., -50°F and the tissue will crumble and become powder. The Ideal tissue should cut like butter, smooth and in one piece.
b) Blade sharpness and angle: The blade should be sharp and should be changed approximately once every 2 weeks. A dull blade cuts dull. Equally important is the blade angle. There is an optimal angle between blade and tissue: • Too steep an angle and the tissue will crumble like it was too cold. • Too shallow, then two things will happen. The section will alternately skip and not cut and then it will cut, but too thick.
