Effusion tumor cell (ETC) detection from cytology samples following long-term storage

Abstract

Abstract Introduction/Objective Circulating tumor cells (CTCs) are shed from solid tumors into blood. The RareCyte CTC system involves multiplexed fluorescence whole-slide imaging of blood for antibody-based identification of EpCAM/cytokeratin-positive and CD45-negative CTCs. We have previously established an assay to detect carcinoma effusion tumor cells (ETCs) from remnant pleural fluid samples employing RareCyte, providing a potential avenue for tumor cell detection from any cytology sample. To facilitate this, our aim is to evaluate slide storage conditions. We hypothesize that long-term storage of pleural fluid ThinPrep slides at -20°C prior to RareCyte analysis will yield similar ETC numbers and characteristics to freshly-prepared slides. Methods/Case Report ETCs were identified and counted via RareCyte according to our previously established criteria (EpCAM mean fluorescence intensity (MFI) > 100 arbitrary unit (a.u.), CD45 MFI < 100 a.u., and cellular morphology). We analyzed 7 paired fresh-stored pleural fluid samples. Stored samples were maintained at -20°C for 154 or 385 days. ETCs counts between the fresh and stored conditions were compared using a Wilcoxon signed-rank test. Marker intensities from the 35 EpCAM-brightest cells across one paired fresh-stored sample were compared using a two-tailed independent t-test. The cutoff for significance is p<0.05. Results (if a Case Study enter NA) The median (25%, 75%) number of cells detected in the 7 fresh and stored samples was 36 (14, 129) and 13 (5, 22), respectively, and was not significantly different (p=0.08). Although the MFIs for DAPI nuclear staining (p=0.12) and CD45 (p=0.82) were not significantly different between the fresh and stored 35 EpCAM-brightest cells, cytokeratin (p=0.02) and EpCAM (p<0.00001) MFIs were significantly less in stored samples. Conclusion Detecting ETCs is possible after long-term storage at -20°C despite a significant decrease in EpCAM and cytokeratin MFIs in stored samples. In this small sample, paired ETC counts were not significantly different. These preliminary results provide a foundation for larger studies to define storage conditions.