Evaluation of pathological tissue specimens using the rapid freezing technique with ethanol

Background: We have been routinely using the rapid freezing technique for making an intraoperative diagnosis. However, the microstructure of the specimens is mostly poor in the examination of frozen sections compared with that in the examination of formalin-fixed paraffin-embedded (FFPE) sections. To assess any improvement, including the histopathological condition of the samples, and cost-effectiveness of the freezing technique, we evaluated and compared the quality of pathological specimens preserved by rapid freezing devices with ethanol (demonstration units) with specimens preserved using a conventional freezing device with acetone as a solvent (control unit). Methods: We created frozen sections using the demonstration and control units and performed quality evaluation of the samples by hematoxylin and eosin staining and immunohistochemical evaluation. Freezing temperature settings for the demonstration units were-16C for the first experiment, - 30C for the second experiment, and - 45C for the third experiment and that for the control unit was-70C. We used eight specimens for the first experiment and 14 specimens for the second experiment with demonstration unit A. For the third experiment, we used 25 specimens with demonstration unit B and 26 specimens with demonstration unit C. Results: Samples frozen at - 16C failed to show non-inferiority (equivalent or superior quality compared to those prepared using the control unit). The percentage of samples evaluated as non-inferior increased to 71 % at -30 C and to 68 % and 65 % at -45C with demonstration units B and C, respectively. Immunohistochemical evaluation was comparable to FFPE sections under any freezing temperature. Furthermore, due to the size of the apparatus and the large amount of ethanol required, the demonstration units were considered inferior to the control unit in terms of safety, convenience, and running costs. Conclusions: At - 30 C or less, 60-70 % of the frozen specimens created with ethanol freezing devices were non-inferior to those produced using the conventional method. However, the devices must be miniaturized for a practical and optimum freezing technique.