Rare cell events may serve as diagnostic, prognostic, and predictive biomarkers of disease. For instance, tumor cells may be shed into peripheral blood long before clinical symptoms develop, metastasis occurs, or cancer recurs.1 Similarly, circulating endothelial cells may be used as markers of tumor angiogenesis, vascular injury, and cardiovascular disease.2 Quantification of residual cancer cells may also be used to predict disease remission following treatment.
Challenges in detecting rare cell events
In all of the above cases, the circulating cells or residual cells are extremely rare events, often representing anywhere between 0.01 and 0.0001 percent of the total sample.2 Given their low frequency, it may be necessary to parse millions of events to obtain a statistically and clinically relevant result. Therefore, important considerations when choosing a technique for rare cell detection include enrichment strategies, speed of detection, and accuracy of detection.
Flow cytometry and fluorescence microscopy have been extensively used to detect rare cell events in blood, bone marrow, and solid tumors. But is one more suitable than the other?
Multicolor flow cytometry offers the advantage of detecting up to 20 fluorescently labeled antibodies, allowing for in-depth analysis of cell types. Furthermore, samples do not necessarily have to be enriched because flow cytometers can efficiently quantify single cells. Most flow cytometers can detect thousands of cells per second. Rare cells can also be sorted and collected for further analysis. However, to reduce the time spent analyzing a large volume of a single sample, enrichment of target cells is often performed.
Positive enrichment involves labeling the sample with tumor cell antigens such as epithelial cell adhesion molecule (EpCAM). During negative enrichment, samples are labeled with hematopoietic cell antigens, such as CD45 for leukocytes. Labeled cells are then separated from the rest using magnetic beads. Alternatively, samples may be enriched using density gradients or microfluidic devices that separate cells based on their physical properties.3 To ensure that cells recovered after flow cytometry can be used for other types of analyses, enrichment protocols need to be chosen such that they do not compromise cell viability.
One of the greatest limitations of flow cytometry is its low resolution, which fails to provide an adequate analysis of cell morphology and therefore visual confirmation of cell identity. Therefore, flow rates and gating strategies may need to be extensively optimized to ensure high target cell specificity to minimize the occurrence of false positives and false negatives.4
Immunocytochemistry is widely used clinically to enumerate and characterize circulating tumor cells. Following EpCAM enrichment, cells are immunostained with a nuclear dye, leukocyte-specific antibodies, and epithelial-specific antibodies. Semiautomated fluorescence microscopes or scanning fluorescence microscopes are then used to identify possible rare cell events based on cell surface marker expression. The system presents computer-generated cellular images to an operator for final review. Therefore, it offers the ability to study cell morphology, viability, and protein co-localization.
However, fluorescence microscopy is limited by the number of fluorophores that can be used to characterize a rare cell population. Imaging is usually restricted to three or four fluorophores at a time. In addition, manual identification of morphological features makes this a subjective and time-consuming process.
The best of both worlds
What if rare cell events could be detected with the speed of a flow cytometer and the spatial resolution of a fluorescence microscope? This can be accomplished with the use of imaging flow cytometry. With the exception of cell sorting, imaging flow cytometry offers all the advantages of regular flow cytometry and provides the added benefit of visualizing morphology at a single-cell level. It is a useful tool in the enumeration of rare cells and their phenotypic characterization because it allows morphological and fluorescent data to be analyzed at both a single-cell and population level.5
Innovations in microscopy and flow cytometry
Traditional flow cytometry and microscopy continue to be updated in order to develop better methods for rare cell detection. One such approach is acoustic focusing cytometry that accelerates event acquisition without compromising data quality. In vivo flow cytometry6 and in vivo confocal microscopy7 have also been developed to noninvasively quantify and characterize circulating cells within blood vessels. These approaches are yet to be reliably applied in a clinical setting.
The decision to use flow cytometry or microscopy for detection of rare cell events usually depends on the downstream applications. Overall, flow cytometry enables rapid quantification of rare cells without providing morphological insights. Conversely, microscopy has low throughput but provides better characterization. Currently, most common approaches rely on epithelial cell markers for enrichment or capture of circulating rare cells using immunomagnetic beads or flow cytometry, followed by fluorescence microscopy or nucleic acid sequencing for further characterization. Emerging evidence suggests that rare cell populations such as circulating tumor cells and circulating endothelial cells have heterogeneous phenotypes. Therefore, methods that allow both enumeration and better characterization of these cells are needed.
1. Marrinucci, Dena, et al. “Fluid biopsy in patients with metastatic prostate, pancreatic and breast cancers.” Physical Biology (2012).
2. Khan, Sameena S., Michael A. Solomon, and J. Philip McCoy Jr. “Detection of circulating endothelial cells and endothelial progenitor cells by flow cytometry.” Cytometry Part B: Clinical Cytometry (2005): 1-8.
3. Ferreira, Meghaan M., Vishnu C. Ramani, and Stefanie S. Jeffrey. “Circulating tumor cell technologies.” Molecular Oncology (2016): 374-394.
4. Hedley BD and Keeney M. “Technical issues: flow cytometry and rare event analysis.” International Journal of Laboratory Hematology (2013): 344-350.
5. Samsel, L and McCoy Jr. JP Samsel, Leigh, and J. Philip McCoy. “Detection and characterization of rare circulating endothelial cells by imaging flow cytometry.” Methods in Molecular Biology (2016): 249-264.
6. Tan, Xuefei, et al. "In Vivo Flow Cytometry of Extremely Rare Circulating Cells." Scientific Reports 9.1 (2019): 3366.
7. Hu, Yuhao, et al. "Monitoring circulating tumor cells in vivo by a confocal microscopy system." Cytometry Part A 95.6 (2019): 657-663.