Frozen section
Troubleshooting artifacts and poor technique

Author: Jessica Wallace (see Authors page)

Revised: 26 July 2017, last major update October 2011

Copyright: (c) 2002-2017, PathologyOutlines.com, Inc.

PubMed Search: Frozen section troubleshooting

Cite this page: Troubleshooting artifacts and poor technique. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/frozensectiontroubleshooting.html. Accessed August 17th, 2017.
Ice crystal artifacts
  • Due to slow freezing of tissue
  • Solution: Freeze fast (flash / snap); the faster the freeze, the smaller the ice crystals, the less tissue damage (best freezing method is arguably liquid nitrogen)
  • Smaller tissues yield less artifact - optimally tissue should be 0.5 x 0.5 x 0.3 cm or less
  • Never freeze fragments larger than the diameter of the chuck
  • Avoid freezing fat around tissue
  • Blot the outer surface of the tissue dry with gauze before making your block
Microscopic (histologic) images

Images hosted on other servers:

Left: ice crystals in edematous stroma by frozen section, right: H&E

Nuclear ice crystals (particularly a problem with thinner sections): left - lung adenocarcinoma, right - uterine sarcoma


Glioma

Breast tissue

Knife artifact
  • A nicked cutting blade will produce a split / tear in your section
  • Solution: change your blade every few cases; some institutions use a new blade for each case
Over / underfreezing
  • Overfreezing can cause section to have holes
  • Solution: polish block with a couple extra turns of the blade to create friction and warm up block by pressing on it with your finger (5 - 10 seconds)
  • Underfreezing can be troublesome for fatty tissue
  • Solution: add heat sink to block or select rapid freeze setting on your cryostat (if available)
Staining issues
  • Dirty "stain line" can cause floaters (extraneous foreign tissue) to adhere to slides; overly diluted stains and alcohols can diminish slide quality
  • Poor staining hinders frozen section diagnoses, as nuclear detail is compromised
  • Solutions: (a) maintain a clean stain line by frequent solution changes; (b) follow recommended staining times; (c) don't rush
  • Note: brain tissue may stain best in eosin for 60+ seconds
  • Water: should be changed after each frozen section
  • Alcohols and stains: change at least weekly, alcohols may need to be changed more frequently depending on work load
Fatty tissue
  • Includes lymph nodes, breast, skin; may be too soft to cut
  • Solution: maintain an extremely cold cutting temperature (-20C)
  • Firm lymph nodes, spleen, brain and liver cut better at -10C; tissue may shatter if sectioning is performed at lower temps
Air bubbles
  • May be trapped under cover slips, which can cause the underlying tissue to dry out
  • Solution: make sure an appropriate amount of resin (2 drops) is applied; gently move air bubbles off the slide with finger or tweezers; do not press on the slide too hard or it will break
Overly thick sections
  • May cause tissue to fall off slide
  • Solution: reduce the cryostat's sectioning thickness