Frozen Sectioning Flashcards
What are the principles of frozen sectioning?
Sample is not processed
Tissue becomes firm by freezing it
Ice crystals provide support for cutting thin sections
Water within tissue is frozen quickly
What are the main reasons for creating slides on the cryostat?
- Stat samples – patient in the OR and surgeon wants a result within 15 minutes. Cut on cryostat and do a rapid H&E. Pathologist examines slide right away and reports to the OR.
- Lipids, etc. - Want to demonstrate lipids or other elements of tissue that may be removed during processing
- Histochemistry
- Fluourescent antibody techniques
What are the advantages of frozen sectioning / cryostat use?
- Rapid diagnosis – eliminate the need to process tissue
2. No shrinkage of tissue
What are the disadvantages of frozen sectioning / cryostat use?
- Structural details poor
- Thicker section
- Ice crystal artifact
- No ribbons
- Biohazard concerns with fresh tissue
How are tissues treated / prepared for cryostat?
TISSUE – FRESH or 10% NB FORMALIN – WASHED!
What precautions must be taken with the tissue intended for frozen sectioning?
Fresh tissues are treated with Routine Practices.
What are the waste removal considerations for frozen sectioning?
All waste must go in biohazard bags and be autoclaved or incinerated.
What kind of embedding medium is used when frozen sectioning on a cryostat?
Water based embedding medium
Name some cryostat parts?
Chuck Holder
Freezing Bar
Anti-roll device (face Plate)
A cryostat is basically a __________ _________ inside a __________.
rotary microtome
freezer
Fresh tissues are usually quickly frozen at what temperature range?
Quickly frozen at - 10°C to -30°C, dependent on the tissue type.
What is brain and lymph node typically frozen at?
Brain and lymph node at -10°C.
What is liver, kidney, and spleen typically frozen at?
Liver, kidney, and spleen at -15degC
What is muscle and thyroid typically frozen at?
Muscle and thyroid at -20C,
What are these tissues typically frozen at?
a) Skin
b) Breast or other tissues with adipose tissue?
a) Skin at -25C,
b) Breast or other tissues with adipose tissue -30C
What are the typical section thickness for cryotomy?
The section is cut at 6 to 10 um thick. Unable to cut any thinner than that.
Describe the cryotomy process?
Single section is cut. You can use the face plate or a tiny paint brush to gently drag the specimen across the cutting surface to prevent it from rolling up. Then you touch a room temperature slide down to the specimen.
What daily maintenance should be done on a cryostat?
- Daily check of temperature before use.
- Regular defrosting and
- Regular disinfection
- Low temperature oil & grease
What safety considerations are required for the cryostat work area (6)?
- Unfixed tissue is potentially infectious
- Gloves, mask, goggles, gown
- Restrict work area
- Restrict # of instruments
- Restrict # of staff
- Pick up debris with gauze dampened with alcohol
What are the basic steps of frozen sectioning?
- Obtain tissue sample
- Prepare tissue holder
- Water based medium
- Rapid freeze
- “Rough in” tissue
- Set anti-roll device
- Fine cutting
- Pick up tissue section
For decontamination can you use sodium hypochlorite?
NO, cannot use Na Hypochlorite
What is used on cryostats for daily decontamination?
Daily decontamination
- 70% Ethanol (solution) – daily cleaning (not necessary to shut down unit)
- New cryostats – UV or ozone
What is used on cryostats for weekly decontamination (if used often)?
Weekly (if used often)
shut off freezing unit and clean thoroughly with something that can kill TB and HIV, HBV, etc.
95% alcohol, Glutaraldehyde vapour or 40 % formaldehyde vapour can be used.
What to do if? Possible reasons/Remedies.
Tissue breaks off the specimen holder.
Causes
- Not enough contact between specimen and holder because of air trap
- Roughing in too much
Remedies
- Use ample amounts of OCT on specimen holder
- Take smaller increments
What to do if? Possible reasons/Remedies.
No tissue cut when drive wheel advanced
Causes - Out of gear - Mechanism frozen after incorrect maintenance procedures - Tissue not frozen solid Remedies - Put pawl back on ratchet wheel - Ensure use of low temperature oil - Refreeze tissue; check temperature of chamber
What to do if? Possible reasons/Remedies.
Tissue sections roll up before going under the anti-roll plate
Causes
Anti-roll plate not parallel to cutting edge or not far enough ahead of bevel edge
Remedies
Adjust anti-roll plate.
What to do if? Possible reasons/Remedies.
Tissue sections stick to anti-roll plate and/or knife
Causes
Anti-roll plate or knife too warm
Grease or debris on anti-roll plate or knife
Remedies
Close lid of cryostat for a few moments to cool
Clean with absolute alcohol then close lid to cool plate and knife
What to do if? Possible reasons/Remedies.
Sections have vertical splits or are scored or shredded
Causes Knife is nicked. Dirt on anti-roll plate Barb of plastic from damaged anti-roll plate Remedies Sharpen knife or move knife over. Clean and then cool Sand edge of the anti-roll plate on 00 emery cloth fastened to a flat board
What to do if? Possible reasons/Remedies.
Tissue sections are wrinkled and folded
Causes
Debris on knife or anti-roll plate
Dull spot on knife
Tissue left on anti-roll plate
Remedies
Wipe with tissue, cool
Sharpen knife or move knife over.
Clean anti-roll plate with absolute alcohol and cool
What to do if? Possible reasons/Remedies.
Cut sections are not as wide as tissue block (compression)
Causes: - Tissue too warm - Dull knife Remedies: - Chill tissue - Sharpen knife
What to do if? Possible reasons/Remedies.
Tissue shatters with fine cracks parallel to the knife edge
Causes: Tissue too cold when cut
Remedies: Check cryostat temperature gauge
What are the major reasons for nicks and chattering?
Nicks are caused by imperfection on the blade. Chattering usually means something is not tight enough.
What are some tips to handle fatty tissue (3)?
- May have to lower the temperature for fatty tissues. 2. Orient specimen so it hits the fat last.
- Need a very sharp blade.
What can cause shattering?
Block may be too cold.
