Flow cytometric characterization and sorting of ultrasound contrast agents

Ultrasound contrast agents consist of microscopically small gas bubbles encapsulated by elastic shells, and are regularly employed in ultrasound blood pool perfusion diagnostics and currently explored as drug delivery vehicles. Successful development of functional ultrasound contrast imaging demands precise and effective microbubble evaluation techniques. We have examined flow cytometry as a method for in vitro quantification, optical characterization and sorting of microbubbles with flexible lipid shell or albumin shell. Flow cytometry provided reproducible side and forward scatter analysis and quantification of gas filled microbubbles enumeration, at various pressure levels and fluid flow speeds. Quantification allowed determination of the static microbubble stability, indicating increased stability at 4°C compared with storage at ambient temperature. Flow cytometry allowed detection of microbubbles in mixture with cell lines or whole blood ex vivo. Furthermore, flow cytometry analysis permitted microbubble sorting by size or fluorescence, allowing evaluation of isolated microbubble populations. We anticipate that flow cytometry will be a reliable technology for analysis of novel modified microbubbles for use in clinical ultrasonography