A morphological study of cooling rate response in normal and neoplastic human liver tissue: cryosurgical implications.

Author(s): Bischof J, Christov K, Rubinsky B

Publication: Cryobiology, 1993, Vol. 30, Page 482-92

PubMed ID: 8252916 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The aim of this study was to identify morphological effects of different cooling rates on 3 neoplastic and 4 normal liver and colon specimens. The fastest cooling rate was represented by immersion in liquid nitrogen (2000 degrees C/min) while all other cooling rates examined (2 to 360 degrees/min) were performed by freezing on a directional solidification stage. All frozen specimens were then stored in liquid nitrogen until fixation by the freeze substitution method, during which ice was replaced with acetone over a slow two day warm-up time course to room temperature. Morphology of frozen specimens was compared to case-matched controls preserved in 3% glutaraldehyde and embedded in resin.

   Summary of Findings:

   The lobular structure of normal liver specimens snap frozen in liquid nitrogen at an estimated cooling rate of 2000 degrees C/min was preserved, although evidence of partial dehydration and artifacts induced by ice formation within both extracellular spaces and sinusoids, and the cytoplasm of hepatocytes were observed. The size of normal hepatocytes was also modestly reduced when compared to glutaraldehyde-fixed specimens. Compared to specimens immersed in liquid nitrogen, normal liver specimens frozen at a slower rate (360 degrees C/min) had smaller sized hepatocytes, poorly defined cell boundaries and nuclei, obvious large transparent extracellular ice crystals that were surrounded by dense fibrillar structures, and smaller ice crystals that were identified by the authors as intracellular in position. In normal liver specimens frozen at a rate of 22 degrees C/min orderly hepatocytes were replaced by swollen sinusoids filled with extracellular ice crystals surrounded by cells that appeared small, dehydrated and dense resulting in radically altered architecture. Normal liver specimens frozen at the slowest cooling rate examined (2 degrees C/min) consisted of dark clumps of small dehydrated hepatocytes scattered between large extracellular ice crystals within the sinusoids. In comparison to normal specimens, tumor specimens appeared resistant to cellular dehydration, displaying less severe architectural damage at cooling rates of 260-22 degrees C/min. The authors hypothesize that the density of the tumor may have had a protective effect on freeze-induced damage.

   Biospecimens

   • Tissue - Liver
   • Tissue - Colorectal

   Preservative Types

   • Frozen
   • Other Preservative

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Morphology	Light microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Glutaraldehyde Snap frozen
   Biospecimen Preservation	Cooling or freezing method/ rate	2 degrees C/min 22 degrees C/min 260 degrees C/min 360 degrees C/min 60 degrees C/min Liquid nitrogen
   Preaquisition	Diagnosis/ patient condition	Normal Hepatocellular carcinoma Metastatic colon carcinoma

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