Detection of effusion tumor cells under different storage and processing conditions
Cancer Cytopathology. 2024;132(5):297-308. DOI 10.1002/cncy.22803.
How to cite
AMA
Libert DM, Zhu Y, Wang A, Allard GM, Cheng-Yi Lowe A. Detection of effusion tumor cells under different storage and processing conditions. Cancer Cytopathol. 2024;132(5):297-308. doi:10.1002/cncy.22803APA
Libert, D. M., Zhu, Y., Wang, A., Allard, G. M., & Cheng-Yi Lowe, A. (2024). Detection of effusion tumor cells under different storage and processing conditions. Cancer Cytopathology, 132(5), 297-308. https://doi.org/10.1002/cncy.22803BibTeX
@article{libert2024detection,
title = {Detection of effusion tumor cells under different storage and processing conditions},
author = {Libert, Diane M. and Zhu, Yili and Wang, Aihui and Allard, Grace M. and Cheng-Yi Lowe, Alarice},
journal = {Cancer Cytopathology},
volume = {132},
number = {5},
pages = {297--308},
year = {2024},
doi = {10.1002/cncy.22803}
}Cytology alone misses many cancers in fluid that collects around the lungs, detecting malignant cells in only 46% to 60% of malignant pleural effusions. An immunofluorescence assay that flags rare tumor cells by their surface markers can raise that yield, but a laboratory can only adopt it if detection survives ordinary slide handling.
Building on an established method for these effusion tumor cells, the team prepared malignant pleural fluid on ThinPrep slides and smears under seven processing and staining conditions, held some at room temperature, and froze others long-term, then counted tumor cells by immunofluorescence under each condition.
Tumor cells were found under every condition, though staining and long-term freezing measurably shifted the marker signals the count depends on.
Key findings
- Effusion tumor cells were detectable under every condition tested. Across malignant pleural fluid from 13 patient samples, the assay identified effusion tumor cells in all seven slide-processing and staining conditions, all three short-term storage conditions, and after long-term storage at −80°C.
- Ethanol-fixed, unstained ThinPrep slides tracked the established air-dried protocol most closely. Diff-Quik and Papanicolaou staining raised CD45 signal and lowered EpCAM signal enough to push some tumor cells past the assay's CD45 cutoff of 100 arbitrary units, whereas ethanol-fixed unstained slides showed no reproducible marker-intensity difference from the reference condition.
- Tumor cells survived long-term storage at −80°C, with fewer cells and a weaker EpCAM signal. Stored slides yielded a median of 13 effusion tumor cells per sample versus 36 in freshly prepared slides (p=.08, not statistically significant), and EpCAM intensity dropped significantly after freezing.
CyteFinder II HT in the methods
“We performed multiplexed fluorescent staining of the sample slides using the RarePlex 0700-MA Staining Kit, and the slides were scanned using the CyteFinder II HT Instrument (RareCyte Inc).”
— Libert et al., Cancer Cytopathology (2024), Methods, “Multiplexed IF staining, imaging, and effusion tumor cell identification”
Why it matters for CyteFinder II HT users
If you are adapting a rare-cell immunofluorescence assay to a new sample type, the practical question is whether detection holds up once the sample leaves its ideal preparation. This study put that question to effusion tumor cells in pleural fluid. The detection step here is whole-slide scanning: after staining, the CyteFinder II HT Instrument imaged each ThinPrep slide across four fluorescence channels, and a tumor cell was called by a fixed rule, nucleated, EpCAM-positive, and CD45-negative, at an intensity cutoff of 100 arbitrary units. Because the scanner reads the entire slide instead of a pre-selected field, the authors could compare marker intensities condition by condition and still recover tumor cells after staining, room-temperature holds, and long-term freezing. For your own work that is the useful part: the imaging step gave a consistent, quantitative readout stable enough to separate a durable preparation from a lossy one. It also marks where the method strains, since Diff-Quik and Papanicolaou staining and −80°C storage shift the very marker signals the call depends on, so morphology has to carry more of the decision.
