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Suboptimal Snap Freezing

Jon Wilson, MD neuropathologist at arkana laboratories
By Jon Wilson, MD

Mar 25, 2022

Freezing Artifact
Figure 1: Frozen section hematoxylin and eosin 200x original magnification

This 50-year-old patient presented with a two year history of bilateral upper extremity muscle weakness. They also complained of muscle aches and cramps, tingling and numbness. EMG showed features of a chronic myopathic process. NCS showed features of severe symmetric axonal sensory peripheral neuropathy. Their home medications included rosuvastatin.

What do the optically clear spaces represent in Figures #1 and #2?

A. Freezing artifact

B. Autophagic vacuoles

C. Glycogen

D. Lipid



Answer: Freezing artifact

The optically clear spaces represent freezing artifact: the formation of ice crystals within the muscle fibers which displace the sarcomeres. Freezing artifact hampers the morphologic evaluation of muscle biopsies.

Typically, muscle biopsies are “snap frozen” by plunging the tissue into isobutene (2 methyl butane) cooled to ~-155 oC by liquid nitrogen. The cooled isobutene transfers the freezing temperature to the tissue quickly as it remains in direct contact with the tissue.

In contrast, freezing the tissue directly in liquid nitrogen does not transfer the freezing temperature as well because it loses contact with the tissue when it “boils” away from the warmer tissue surface.

A standard cryobath bath used for intraoperative frozen sections does not cool the muscle biopsy down quickly enough, and produces significant freezing artifact.

Freezing artifact can be partially corrected by briefly (but completely) thawing the biopsy tissue followed by re-freezing in liquid nitrogen cooled isobutane.


Reference(s)/Additional Reading

Nix JS, Moore SA. What Every Neuropathologist Needs to Know: The Muscle Biopsy. J Neuropathol Exp Neurol. 2020 Jul 1;79(7):719-733. doi: 10.1093/jnen/nlaa046. Erratum in: J Neuropathol Exp Neurol. 2021 Mar 22;80(4):387. PMID: 32529201; PMCID: PMC7304986


Quick note: This post is to be used for informational purposes only and does not constitute medical or health advice. Each person should consult their own doctor with respect to matters referenced. Arkana Laboratories assumes no liability for actions taken in reliance upon the information contained herein.