Abstract 2380: Detecting human circulating endothelial cells using the 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. In cancer research, CECs have 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. In healthy individuals, CECs are reported to be present in very low numbers: 0.01%-0.0001% of all peripheral blood mononuclear cells. Flow cytometry offers the necessary collection and analysis capabilities for detection of CECs, but is subject to numerous technical challenges. In comparison to traditional hydrodynamic-focusing cytometers, the Attune® Acoustic Focusing Cytometer, with its fast acquisition times and increased precision, overcomes the technological hurdles involved in analyzing CECs. Here we present a method for detecting human circulating endothelial cells using the Attune® Acoustic Focusing Cytometer. The method includes a number of conventional ways to improve rare event detection: a blocking step, a viability stain (SYTOX® AADvanced™ Dead Cell Stain), and the use of a dump channel to eliminate unwanted cells and decrease background fluorescence. The challenge of collecting a large enough number of events in a reasonable amount of time is met by using a collection rate of 1000 µL/min with the Attune® cytometer. 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. Furthermore, this method delivers additional time savings by eliminating wash steps to avoid sample loss and employing a simpler sample preparation method; details and specific panels designed for both the blue/violet and blue/red Attune® cytometer configurations are outlined. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2380. doi:1538-7445.AM2012-2380