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HTO 130

Cryostat/Frozen Sections

QuestionAnswer
The Cryostat is a refrigerated chamber containing what type of microtome? Rotary
For the Cryostat the blade needs to be _________? Free of defects and needs to be sharp
How are the sections picked on a cryostat slide? Sections are picked on a WARM slide
For the Cryostat all blades, brushes, disc, chucks, bars, and heat extractor need to be _________? And cleaned with _________? Need to be free of tissue waste And cleaned with 100% Alcohol and gauze.
What can be kept in a specimen container in the cryostat, so it is accessible at all times? 100% Alcohol
The temperature for most tissue in the cryostat is typically at _______. -20C
What is cut in the cryostat at -10C temperature (warmer)? Brain, liver, spleen, lymph nodes, and endometrial scapings
What is cut at -25C temperature (colder) in the Cryostat? FAT
What happens in the cryostat if the tissue splits or fragments? The temperature is too low.
What happens in the Cryostat if the tissue collects at blade edge? The temperature is too warm.
What do you do if the tissue is not sectioning for the cryostat? Make sure blade is sharp and temperature is appropriate for tissue.
What assists freezing tissue quicker and helps to flatten the tissue on the disc? Heat Extractor
what is Freeze Thaw Artifact? Slow freezing of tissue will allow water to form artifacts or holes in the tissue that are ice crystals.
Muscles are frozen with? Isopentane suspended in liquid nitrogen.
What temperature do you store muscles? Store tissue in freezer of -70C.
When using an antiroll plate, the plate has to be ______? Plate has to be parallel to and very slightly above the blade.
For the maintenance of the cryostat the tissue shaving is disposed where? Biohazard bag
What must you do daily for the Maintenace of the cryostat? daily cleaning and wiping chamber with 100% alcohol.
What must you do weekly for the maintenance of the cryostat? Decontaminated weekly typically at the end of the day on Friday.
What must we be sure to have a preventative maintenance plan for the cryostat? Defrosting the machine.
Method of processing rapid diagnosis? (Note: As well as the staining technique does not work on routinely processed tissue) Frozen Sections
Why are frozen sections used? Used for enzyme and immunohistochemical techniques.
What stains require sections of tissue to be cut on the cryostat? why? Oil Red O and Sudan Black B because fat will dissolve in most fixatives except osmium tetroxide.
What forms when tissue is thawed? Ice crystals (which can result in holes in the frozen section.)
What are the greatest hazards in cutting unfixed tissue? Blade and tuberculosis
Tuberculosis can be aerosolized with? Quick Freeze
We do not use quick freeze for? Lung specimens
Cryostat troubleshooting: Freezing artifact. What is the cause and how do you prevent it? Cause: Improper freezing of the tissue. Prevent by: use heat extractor, freeze with isopentane chilled at -150c or ensure tissue is not immersed in saline prior to freezing. (This is a primary problem)
Cryostat troubleshooting: Block loosens or detaches from Chuck while sectioning. What is the cause and how to prevent it? Cause: Chuck too cold when embedding medium was applied. Prevent by: Reattaching tissue to block to clean chuck, avoid storing chucks with medium in cryostat overnight, or avoid storing chucks without embedding medium in cryostat
Cryostat troubleshooting: Tissue not embedded flat on the chuck. What is the cause and how to prevent it? cause: Tissue was not properly pushed down on the chuck Prevent by: use heat extractor to flatten out tissue Note: fix immediately to reduce sectioning deeper into the block and very important to not waste parts of the tissue.
What degree is the tilt of the blade edge? optimum tilt 30 degrees between the center line of the blade and block base
how is the cryostat cooled? by a mechanical refrigeration unit
Frozen sections that are impossible to obtain with a clinical freezing microtome can be obtained using what instrument? cryostat
Most anti roll devices are made for this commonly used blade for the cryostat. high profile blade
What can cause sectioning problems and cause sections to curl when cutting? improper adjustment causes sectioning problems roll plate can cause sections to curl
example of freeze thaw artifact that is disturbed in some tissue. skeletal muscle
tissue may be frozen more rapidly by using what methods? 1.heat extractor attachment 2. dry ice 3. hydrofluorocarbon refrigerants such as freon spray 4. beaker of isopentane suspended in liquid nitrogen
What is the preferred method for freezing skeletal muscle biopsy specimens? Isopentane suspended in liquid nitrogen
What temperature must the isopentane be before introducing the specimen such as skeletal muscle biopsy on which enzyme studies are needed to reduced artifact.? reach the temperature of -150 degrees Celsius before introducing the specimen.
why is liquid nitrogen used alone? because of gas bubbles from around the tissue and they implead on freezing
What must you not leave tissue in the cryostat? Severe dehydration (Freezing and sectioning tissue should not be stored unprotected in the cryostat for any length of time because it will lead to severe dehydration.
If a specimen must be stored frozen for any length of time, what should you do? It should be wrapped carefully to exclude air and to be stored in a freezer at -70 degrees Celsius.
to work with tuberculosis in the cryostat (which is not recommended) what must you use? A negative pressure chamber connected to a higher efficiency HEPPA filter.
What are two hazards for frozen sections? 1. isopentane is extremely flammable 2. people are not used to the cold temperature (frost bite)
the tissue is not embedded flat on the chuck. How do you prevent this? place tissue on a slide surrounding embedding medium & place glass slide on freezing bar of cryostat. Embed medium begins turning white, coat specimen chuck w/ embedding medium over tissue. Let freeze then remove slide by warming slightly with finger.
The specimen given to you at different widths cannot lay flat on chuck. how do you prevent this? Use heat extractor to flatten it. example would be a skin legion.
Note1: Tissue may be frozen in cryostats, process is slow, ice crystals form when tissue is thawed which gives holes. Does not interfere w/ most tissues which rapid diagnosis is needed. Skeletal muscles frozen in this way for subsequent enzyme techniques Note2:large hole sections are worthless for diagnostic studies. Size& amount of ice crystals formed is directly proportional to speed of freezing tissue. Heat extractors are added to some cryostat, but frozen sections made by isopentane & liquid nitrogen
Created by: Natasha Staley
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