Immunocytochemistry is an advanced diagnostic tool used to identify the origin of tumor cells. The present study aimed to clarify the usefulness of cryopreserved, air-dried cytology samples for the detection of cytokeratin and vimentin. Air-dried smear samples were prepared from canine tumors and stored at –30°C or room temperature without fixation. The duration of cryopreservation varied from 2 months to 4 years and 8 months. Formalin-fixed paraffin sections were also prepared from these tumors. These samples underwent enzymatic immunocytochemistry and immunohistochemistry for the detection of cytokeratin and vimentin. Immunoreactivity for cytokeratin was detected in samples cryopreserved for a maximum of 3 years and 10 months. Immunosignals for vimentin were clearly detected for 2 years. Expression of cytokeratin and vimentin was not detected in samples stored at room temperature for 1 week. Immunoreactivity was observed in all specimens using immunohistochemistry. These findings suggest that immunocytochemistry for cytokeratin and vimentin can be safely applied to air-dried smears that have been cryopreserved at –30°C for at least 2 years. This simple method may be useful for retrospective cytological studies and/or re-evaluation of cytology results.
Immunocytochemistry (ICC) is an advanced technique that can aid in identifying the origin of malignant cells in cases of carcinoma, sarcoma, and lymphoma (Camus et al. 2020; Raskin et al. 2019; Sawa et al. 2015, 2017, 2018). The detection of cytokeratin as an epithelial marker and vimentin as a mesenchymal marker is particularly useful because they can be used to differentiate between the origins of epithelial cells and nonepithelial cells, respectively (Camus et al. 2020; Raskin et al. 2019; Sawa et al. 2017, 2018). ICC is usually performed when distinguishing the tumor origin is difficult using conventional cytological methods involving Romanowsky stains. Therefore, stored non-stained smears are often inevitably used for ICC after the standard diagnosis. Preservation of antigenicity in stored smears is the most important challenge for ICC. A simple method for preserving samples is therefore required, especially in veterinary clinics that do not have laboratories or clinicians with expertise in ICC.
Cryopreservation is the most common method used for long-term storage of paraffin sections and frozen sections for immunohistochemistry (IHC) (Wester et al. 2000; Ohta et al. 2008). This method is presumably applied empirically in many clinicopathological laboratories and can be easily adopted in most veterinary clinics. However, there is no evidence regarding the accuracy and reliability of ICC results from unfixed air-dried smears stored in the freezer. Therefore, in the present study, we performed ICC for cytokeratin and vimentin using cryopreserved smears. We aimed to investigate differences in the immunosignals of cryopreserved samples and formalin-fixed paraffin-embedded (FFPE) tissues for IHC.
The present experiments were conducted in accordance with the Guidelines for Veterinary Clinical Research of Kagoshima University in Japan (No. KVH190001). All samples were surgically removed, fixed in formalin, and subjected to histopathological analysis by veterinary pathologists with no prior knowledge of the cytological results. Histopathological analysis revealed a diagnosis of epithelial or mesenchymal tumor in all cases.
The following criteria were used to select random tissue samples from dogs hospitalized at Kagoshima University Veterinary Teaching Hospital in Japan: 1) clear histopathological diagnosis of epithelial or mesenchymal tumors, 2) cytological diagnosis corresponding to the histopathological diagnosis, and 3) a large number of epithelial or mesenchymal tumor cells with adequate cytomorphology on smear slides. Exclusion criteria were as follows: 1) clear histopathological diagnosis of non-neoplastic masses, round cell tumors, or tumors of unknown origin; 2) multiple populations of neoplastic cells in histological and cytological tissues; and 3) poor cytomorphological quality in cytological samples.
According to these criteria, smear slides were prepared from neoplastic tissues removed from 16 dogs. Smears obtained from 14 dogs were completely dried in air, following which they were cryopreserved at − 30°C within 24 hours after sampling. The duration of storage varied from 2 months to 4 years and 8 months. Prior to ICC, slides were removed from the freezer and immediately and thoroughly dried with a hair drier using the cold setting. The remaining two samples (one epithelial tumor case and one mesenchymal tumor case) were intentionally left at room temperature (RT) for 1 week in a non-air-conditioned room.
ICC for cytokeratin and vimentin was performed in accordance with a previously described method (Sawa et al. 2017). The primary antibodies included mouse monoclonal anti-cytokeratin (clone AE1/AE3, ready‐to‐use; Dakocytomation, Glostrup, Germany) and mouse monoclonal anti‐vimentin antibodies (clone V9, 1:100 dilution; Thermo Fisher Scientific, Fremont, CA, USA). These primary antibodies have been confirmed to be cross-reactive for canine tissues (Fant et al. 2004; Sawa et al. 2012; Ramos-Vara et al. 2016). Biotin‐labeled horse anti‐mouse IgG (1:200 dilution; Vector Laboratories, Burlingame, CA, USA) was used as a secondary antibody. The samples were then treated with peroxidase-labeled streptavidin (KPL, Gaithersburg, MD, USA), and all antibodies that did not include anti-cytokeratin were diluted in a mixture of 0.25% casein/10 mM phosphate‐buffered saline (PBS). This same casein/PBS mixture was used as the blocking solution. Reactivity was evaluated using 3,3′‐diaminobenzidine (DAB) chromogen (DAB‐buffer tablet; Merck, Darmstadt, Germany) using normal mouse IgG (Dakocytomation) as a negative control. For ICC, the smear slides were completely dried using the cold setting of a hair dryer. Following fixation with cold acetone for 1 min, slides were washed with PBS for 10 s, blocked with 0.25% casein/PBS for 10 min at RT, and incubated with the primary antibody for 10 min at 37°C. After sufficient washing with PBS, the slides were incubated with secondary antibody for 10 min at 37°C and washed with PBS, following which they were incubated with peroxidase-conjugated streptavidin for 10 min at 37°C. After washing with PBS, samples were stained with DAB for 5 min to examine immunoreactivity. The reaction was terminated with cold distilled H2O, and slides were counterstained with Carrazi’s hematoxylin.
The primary and secondary antibodies mentioned above were also used for IHC, which was performed using the same methods as ICC, except that the antibodies for cytokeratin and vimentin were incubated for 20 min at RT, the secondary antibody was incubated for 30 min at RT, and peroxidase-conjugated streptavidin was incubated for 30 min. Immunoreactivity was visualized using DAB with normal mouse IgG (Dakocytomation) as a negative control.
In the present study, all smears and sections were evaluated as positive (+) or negative (-) for immunoreactivity using light microscopy. Signal strengths were compared between ICC samples and negative controls. The ICC signals were also compared with the standard IHC signals.
Specific ICC signals were observed in 12 of 16 cases (Table 1). Immunosignals for cytokeratin were clearly detected with ICC in seven of nine frozen-stock specimens that were diagnosed as epithelial tumors. Both cases cryopreserved for 4 years and 8 months were negative for cytokeratin in ICC. Moreover, cytokeratin staining was not observed in the sample stored at RT for 1 week. Immunosignals for vimentin were clearly detected with ICC in all five frozen-stock specimens that were diagnosed as mesenchymal tumors. On the other hand, one case stored at RT was negative for vimentin with ICC. Examples of the cases are shown in Figs. 1 and 2. Figure 1 shows a freeze-stock sample that was stored for 3 years and 10 months. Clear immunoreactivity for cytokeratin with good-quality visualization was obtained in the tested samples using ICC (Fig. 1b). The freeze-stock sample stored for 4 years and 8 months was negative for cytokeratin in ICC but was positive in IHC (Fig. 2). In ICC, we observed clear immunoreactivity for vimentin with good-quality visualization in samples cryopreserved for 2 years. No immunoreactivity for cytokeratin or vimentin was observed in either of the specimens stored at RT. We were also unable to detect the expression of vimentin in non-malignant mesenchymal cells, such as macrophages, on the same slide in the cases diagnosed as a mesenchymal tumor. However, immunoreactivity was observed in both of these cases in the IHC analysis.
The present study aimed to investigate the usefulness and limitations of ICC staining when using cryopreserved cytological specimens. To achieve this aim, we compared the immunoreactivity signals obtained via ICC and IHC. Stable cytokeratin and vimentin staining was observed in samples that had been cryopreserved for less than 2 years. However, detection of cytokeratin and vimentin was difficult in samples stored long-term at RT.
Valli et al. reported that formalin-fixed preservation provides long-term stability. However, their preservation method requires antigen retrieval using a heat-induced epitope involving citrate buffer (pH 6.0). Moreover, antigens, especially for lymphocyte subtyping, are not active after formalin fixation (Valli et al. 2009). Therefore, formalin is not always suitable for use in a fixative solution for ICC. In our study, we used cold acetone, as previously reported (Sawa et al. 2017). Sawa et al. examined multiple fixative solutions in their study and revealed that acetone provided sufficiently strong signals without antigen retrieval (Sawa et al. 2017). Cryopreservation without fixation may therefore broaden the range of target molecules and antibodies.
Sauer et al. investigated the effects of cryopreservation on immunoreactivity in liquid-based cytological specimens stored at − 20°C or − 74°C. They found no reduction of immunoreactivity for the estrogen receptor or progesterone receptor after 3 and 6 months of storage (Sauer et al. 2010). However, their method requires a preservation solution for liquid-based cytological specimens. In contrast, immunoreactivity for cytokeratin and vimentin remained stable over a long period of time without the need for fixation, antigen retrieval, or preservative solutions in this study. We stored our specimens at − 30°C. However, an ultra-low temperature freezer may enable longer-term storage of specimens than that used in our study.
In this study, we only examined samples stored at RT for 1 week. Despite this short observation period, immunoreactivity was lost. Usually, however, 1 week at RT is not long enough for complete loss antigenicity. Raskin et al. reported storage of samples at RT for a longer period (20 weeks) without loss of signals (Raskin et al. 2019). The present study was conducted during the summer in Japan, during which temperatures and humidity are high. Furthermore, our facility is located in an area that is even hotter and more humid than most areas. The RT samples were intentionally stored in a non-air-conditioned room, and our results suggest that such a harsh storage environment can lead to early loss of antigenicity depending on the temperature and humidity. Thus, we recommend that remaining smears should be cryopreserved, even if only for a short time.
This study had some limitations. First, we did not evaluate specimens stored at RT in a temperature-controlled environment, in the refrigerator, or in a home freezer. Second, the number of cases was limited. Third, we limited our investigation period for vimentin immunoreactivity to 2 years. Detection of vimentin may therefore be possible beyond 2 years. Further studies are required to clarify preservation stability at different temperatures using a larger number of samples and in different species. Lastly, home freezers are common in most veterinary clinics, especially primary facilities. These freezers do not allow for strict temperature control, and constant fluctuations in temperature may occur due to defrosting. Moreover, these freezers store samples at a much higher temperature (approximately − 20°C) than that used in our study. Therefore, the results of the present study may not simply be applicable when using a home freezer.
In conclusion, cryopreservation of immunocytochemical specimens is a simple and convenient method that can be performed without any special equipment. This method may be applicable to staining for other antigens and may aid in the retrospective analysis of cytological specimens or in confirming diagnoses.
The authors declare that no funds, grants, or other support was received during the preparation of this manuscript.
The authors declare no conflicts of interest associated with this manuscript.
All authors contributed to the study’s conception and design. Material preparation, data collection, and analysis were performed by Yu Furusawa, Mariko Shima-Sawa, Tatsuro Hifumi, Noriaki Miyoshi, Osamu Yamato, and Akira Yabuki. The first draft of the manuscript was written by Yu Furusawa, and all authors commented on previous versions of the manuscript. All authors have read and approved the final manuscript.
All data included in this study are available on request from the corresponding author.
This research was performed in accordance with the Guidelines for Veterinary Clinical Research of Kagoshima University in Japan (No. KVH190001).
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Table 1 is available in the Supplementary Files section