To evaluate FCM in comparison to standard frozen sectioning, that often yields results of subpar quality in specimen rich in adipose tissue, with structural changes that might prevent correct diagnoses, we compared FCM with standard in-house procedures for frozen sections. This study was driven by two main intentions. The one aspect was that we wanted to investigate the performance of FCM in specimens that are difficult to process in frozen sections. Another goal was assessing the suitability of FCM for rapid intraoperative telepathology which is of considerable interest for institutions without an in-house pathology lab. Many tumors are embedded in adipose tissue and a meaningful analysis regarding their size and exact location may not be possible in frozen sections in some cases since cryotome sectioning often leaves empty spaces in areas of adipose tissue.
Diagnostic accuracy and applicability of FCM has been shown to be excellent in a variety of studies: Bertoni et al showed that diagnostic accuracy and learning curves were very good for pathologists in their study with prostatic tissue (7). Schüürmann et al also showed a high correlation between the digital images and the H&E-stains in their study on skin samples (12). Our study confirmed the very good diagnostic accuracy for detecting malignancy (16 out of 17 cases yielded the same result compared to H&E analysis. The only discordant case was scanned in different orientation and therefore did not depict the relevant tumor area. Due to the H&E-like pseudo-staining of the images, interpretation was easy and intuitive for the pathologists.
A very promising perspective of FCM is intraoperative telepathology, which is gaining increasing relevance as pathology departments around the world are moving towards fewer, more specialized diagnostic centers (13) and considering the shortage of pathologists in rural areas that is likely to increase in the future: Tissues can be processed in one institution that does not require an on-site pathology department or equipment and can then be analyzed in a remote specialized center that theoretically can be located anywhere in the world.
A relevant aspect that needs to be assessed when considering FCM as an alternative to frozen sections is the duration of the procedure. The staining and scanning procedure with the VivaScope® 2500M-G4 was 4-6 minutes and thus comparable to an average frozen section. The whole procedurre is entirely carried out by one person.
The tissue samples submitted for frozen section analysis are typically first assessed by the pathologist and then transferred to a lab technician for freezing, sectioning and staining. The tissue processing is carried out by a skilled pathologist/technician-team typically and requires at least 4-6 minutes. In case of adipose tissue or other specimens that are difficult to process in frozen section histology this timeframe might be far exceeded.
While scanning time can vary based on sample size and settings, the entire procedure was usually at least comparable and in cases with very small samples slightly faster than the average duration of frozen section.
As shown above the quality of the scanned tissue samples is comparable to the frozen sections but is inferior to that of FFPE sections especially in terms of cytoplasmic details and structure of extracellular matrix.
The loss of tissue for subsequent analysis due to frozen sections can put the pathologist in a significant predicament in cases when diagnosis from paraffin-embedded material without frozen section artefacts is required and the frozen section is needed for intraoperative guidance of resection. An important benefit of FCM is that no tissue is lost due to sectioning and the entire specimen remains available for subsequent analyses which is of particular importance for small samples. In addition tissue does not get frozen and can therefore be treated like a normal specimen in subsequent analyses without the risk of freezing artefacts. Mechanical alterations of the tissue introduced by the scanning process were not detectable and AO staining did not affect subsequent processing for conventional FFPE histology. We however did not assess a potential impact of tissue processing for FCM on molecular pathology analyses.
We found that FCM is an efficient and promising alternative to frozen sections especially if specimens containing adipose tissue. It adequately portrays areas of interest (tumor areas, adipose tissue) and correct assessment of presence or absence of tumor was possible in 16 out of 17 cases with one case being scanned with insufficient quality to perform an adequate diagnosis.
One of the drawbacks of FCM is that depiction of extracellular matrix is somewhat different to the pattern seen in H&E histology, which might, in certain tissues, obscure the interaction of tumors with the surrounding stroma. In our study, we however did not experience incorrect assessment of tumor invasion or the lack thereof. Further research into adequate depiction of extracellular matrix should be conducted in future studies in order to achieve an even closer match of FCM images with H&E histology. Another limitation of FCM that was more relevant for our study is the representation of resection margins: We sometimes observed ill-defined borders of the specimens most likely due to lifting of margins. The inability to visualize ink applied to the specimen’s surface for marking resection margins also complicated the exact measurement of the distance of tumor cells from the resection margin. This might be a serious issue in cases where the tumor is very close to the margin.
In conclusion, we found that when comparing frozen sections to FCM images generated with the VivaScope® 2500M-G4, FCM images had greater tissue integrity, morphological detail and an adequate quality of staining not only in regards to adipose tissue but also tumor areas with a weakness in depicting the sample margins. Despite this drawback, the VivaScope® 2500M-G4 has the potential to expand the application of rapid tissue analysis as speed and quality potentially exceeds that of frozen sections especially in tissues rich in adipose cells as is the case of breast tissue.