For diagnostic purposes, cryofixation of tissues is a daily routine technique to investigate rapidly about the presence of tumours during a surgical procedure in patients. The tissue is placed without cryoprotectant in liquid isopentane, or in liquid nitrogen or in contact directly with a cooled metal block inside the cryostat used for cutting and preparing the specimen. It is well known that severe damages are produced during freezing, principally caused by intracellular ice formation, but until now these studies are performed qualitatively, without any quantitative approach. In order to obtain quantitative indexes we have performed a morphometric analysis (local fractal dimension and Euclidean indexes) of cryofixed muscular tissues according to the different techniques. Ninety-six microscopic fields, corresponding to about 1000 muscle fibres and 1493 nuclei, were automatically examined. After freezing, large voids inside the cells (ice-.tissue interfaces) and some cracks among the fibres (due to shrinkage of the cells) were present. Liquid isopentane or liquid nitrogen produced a statistical increase of the complexity value of the histologic images (quantization of the ice-tissue interfaces, p<0.001) respect to the formalin-fixed samples (“gold standard”), while cryofixation performed inside the cryostat chamber at t = -20°C produced a complexity value close to the formalin-fixed samples. Shrinkage of the muscle fibres was higher in the samples cryofixed inside the cryostat chamber (p<0.001). Cryofixation inside the cryostat at t = -20°C or by liquid nitrogen caused decrease of the nuclei dimensions and altered nuclear morphology (p <0.01), while liquid isopentane appeared not affecting the nuclei of the fibres.
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