Detecting Rare Events

Identify Circulating Endothelial Cells With the Attune® Acoustic Focusing Cytometer

Circulating endothelial cells (CECs) are mature cells shed from blood vessel walls during the natural process of endothelial cell turnover. Elevated levels of CECs have been reported in a host of pathological conditions, including cardiovascular disorders, infectious diseases, immune disorders, post-transplantation analysis, and cancer [1]. In cancer research, the level of CECs has been suggested as a noninvasive biomarker for angiogenic activity, providing insight into tumor regrowth, resistance to chemotherapy, early recurrence, and metastasis formation during or after chemotherapy [2]. In healthy individuals, CECs are reported to be present in very low numbers: 0.01%–0.0001% of all peripheral blood mononuclear cells [3].

Detecting CECs With the Attune® Cytometer

Flow cytometry offers the necessary collection and analysis capabilities for detecting CECs, but it is subject to numerous technical challenges. With fast acquisition times and increased precision, the Attune® Acoustic Focusing Cytometer overcomes the technological hurdles involved in analyzing CECs with traditional hydrodynamic focusing cytometers. The Attune® cytometer precisely aligns cells using acoustic forces independent of the fluid stream, delivering much higher throughput than traditional flow cytometry.

To detect CECs with the Attune® cytometer (Figure 1), we collected a large number of events in a relatively short time by using a high-speed collection rate of 1,000 µL/min. This setting enables the collection of more than 4,000,000 live white blood cell (WBC) events in just 35 minutes; the acquisition time using a traditional hydrodynamic focusing cytometer would be 10–12 times longer, close to 6 hours.

An additional obstacle when detecting rare events is cell loss during sample processing. For detecting CECs, we used a simplified lyse, no-wash procedure to significantly reduce the possibility that cell subpopulations would be affected during sample preparation, while also offering considerable time savings. Furthermore, we employed several conventional methods to improve rare-event detection: a blocking step, the fluorescent SYTOX® AADvanced™ Dead Cell Stain, and the use of a dump channel to minimize unwanted cells and decrease background fluorescence.

Detecting CECs with the Attune® cytometer  
Figure 1.  Detecting CECs with the Attune® cytometer. The Attune® Acoustic Focusing Cytometer was employed for acquisition of 8.6 million total cells in normal lysed human blood and for detection of the rare CEC population (representing the CD31+/CD34+ cells in the upper-right quadrant) using Molecular Probes® flow cytometry reagents. 


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