Procedure for Frozen Sections

procedure^[1]

1. Check that all needed materials are available (within easy reach) and at the correct temperature:
   • Inside cryostat (cold): microtome blades, small and large brushes, forceps
   • Outside cryostat (warm): OCT, positively charged slides, Kimwipes, gauze, applicator sticks, clean metal disks (chucks), pencils/solvent resistant markers, coplin jar with 10% NBF
2. Receive and accession fresh specimen in frozen section log book according to procedure for Accessioning.
3. Record cryostat temperature daily.  Ensure temperature is set appropriately for the tissue you are cutting (routine = -20ºC)
4. Place clean chuck in the cryostat quick-freeze shelf.  Put an even layer of OCT medium on the base of the chuck and allow to freeze, ensuring there are no bubbles present.
5. While OCT is freezing, label the required number of positively charged slides with the FS accession number, patient name, block and slide number, and your initials.  Label one extra backup slide in case it is needed (the slide number can be left blank and added after picking up section).  Keep these slides clean on a Kimwipe within easy reach outside of the cryostat until needed.
6. When OCT base is frozen, don appropriate PPE (gloves and eye protection).  Remove chuck from cryostat, add a thin layer of OCT on top and orient the tissue specimen appropriately.
7. Place chuck and specimen back onto the quick freeze shelf.  Layer just enough additional OCT around the specimen to cover and support the sides and top of the tissue.
8. For tissues that do not require a specific orientation, the weighted heat extractor may be  used to facilitate rapid freezing once the top layer of OCT has turned white and begun to solidify (do not place directly on fresh tissue as it will distort tissue morphology). Freezing sprays create aerosols and are not recommended, especially for lung tissue (Covid-19, TB risk)
9.  Using the control panel, retract the chuck holder away from the knife assembly.
10. Place frozen tissue + chuck into the chuck holder and tighten to secure.
11. Carefully install a new microtome blade in knife assembly, as per routine microtomy procedure.  Ensure blade is locked using the lever and immediately put up the knife guard.
12. Unlock the drive wheel of the microtome and lower the chuck to line up with the blade height.
13. Using the control panel, advance the chuck with the tissue to line up just behind the knife edge.
14. Depending on the size/thickness of tissue, adjust the micrometer setting to approximately 20 µm for trimming (less for small specimens).
15. Rough trim the specimen until the full face is visible.
16. Adjust micrometer setting to 5 µm (or as required for staining method to be performed)
17. Polish the cutting surface by cutting 2-3 sections.  Discard onto waste tray using cold brush and gently clean debris from around block and knife assembly.
18. Using a slow steady motion, cut the next section and use the cold small brush to catch the leading edge of the OCT.  Guide section gently down the face plate as it comes off of the knife, smoothing it out if necessary. (Alternatively: the anti-roll plate may be adjusted parallel to and just above the knife edge to guide and flatten the section onto the face plate, then carefully moved aside for step 19)
19. Take the first labelled slide and holding it steady, line it up with the section on the face plate.  Gently lower it in one smooth motion so that the section adheres to the slide.
20. Put up the knife guard.
21. Assess the section for quality.  If acceptable, immediately place slide in coplin jar of 10% NBF for 1 minute to fix.
22. Repeat from step 18 for subsequent sections/slides as required.
23. Gently rinse slides in coplin jar of distilled water and proceed with requested staining methods.
24. Clean all debris from inside cryostat onto waste tray.  Fresh tissue debris must be collected in red anatomical waste bags for disposal.
25. Cryostat and instruments are to be disinfected routinely.