Single versus coincidence detection of cell-derived vesicles by flow cytometry
||E. van der Pol, M.J.C. van Gemert, A. Sturk, R. Nieuwland, T.G. van Leeuwen|
||SPIE Photonics West 2013, San Francisco, United States|
||February 3, 2013|
||San Francisco 2013 Coincedence detection.pdf (1,859 kB)|
Body fluids contain cell-derived vesicles (<1 μm) which are clinically relevant. The most common method to detect vesicles is flow cytometry. We established the hitherto unknown relationship between light scattering and the vesicle diameter by combining light scattering measurements of beads with Mie calculations. We showed that vesicle detection by flow cytometry relies on two mechanisms: (1) detection of single, relatively large, vesicles scattering more light than the detection limit; and (2) coincidence detection – i.e. multiple relatively small vesicles are simultaneously illuminated and counted as a single event. Coincidence detection allows the detection of smaller vesicles than previously thought possible.
Background: Body fluids contain cell-derived vesicles ranging from 30 nm to 1 μm in diameter. The function, origin, and composition of these vesicles is disease dependent and therefore vesicles contain clinical information. The most common method to detect vesicles is flow cytometry, which guides vesicles through a laser beam in a hydrodynamically focused fluid stream. The unknown relationship between the measured light scattering intensity and the vesicle diameter resulted in unexplained contradictions between expected and observed results.
Methods: We combined light scattering measurements of polystyrene and silica beads with Mie calculations using an estimated refractive index of vesicles.
Results: We established the relationship between measured light scattering and the diameter of vesicles. We show that common gating strategies based on beads select vesicles (and cells) ranging from 800 to 2400 nm in diameter. For our flow cytometer, the smallest detectable silica beads were 204 nm, corresponding to vesicles ranging from 300 to 700 nm in diameter. However, we demonstrated that multiple vesicles <220 nm or multiple 89-nm silica beads were counted as a single event at sufficiently high concentrations.
Conclusions: Vesicle detection by flow cytometry relies on two mechanisms: (1) detection of single, relatively large, vesicles that scatter more light than the detection limit; and (2) coincidence detection – i.e. multiple relatively small vesicles are simultaneously illuminated by the laser beam and counted as a single event. Coincidence detection allows the detection of smaller vesicles than previously thought possible and explains why flow cytometry underestimates the concentration of vesicles typically 1,000-fold.