A high volume parallel acoustic flow cytometer for the detection of rare cells or particles in a large sample volume with a low background concentration (i.e. very dilute samples).

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 DESCRIPTION (provided by applicant): Many biomedical applications require the detection of rare cells or particles in large sample volumes. These applications can be divided into those that require the detection of highly dilute cells in a large sample volume and those that require the detection of rare cells in a high background concentration of similar cells within a large volume. Though flow cytometry is the gold standard in the detection of cellular populations, its volumetric sample delivery rate dramatically limits its use for large volume samples and its analytical rate prevents its use for these applications. However, recent work funded by the NIH has resulted in several advances that can be used to develop a high volume flow cytometer that will be of immediate use for high volume rare cell detection in low cell backgrounds. Using a synergistic combination of parallel acoustic flow cells, a cutting edge high speed sCMOS camera, and a novel optical configuration, our high volume parallel acoustic flow cytometer (HVPAfc) will collect multicolor flow cytometry data at analytical rates of 100,000 cells per second and sample delivery rates of 50 mL/minute. This instrument will have immediate value for many applications, such as the detection of bladder cancer cells in urine or pathogenic bacteria in milk or water, and thus will have a large impact on the field and the marketplace. To create the GenI HVPAfc, we will first construct an acoustic flow cell that creates many parallel sample streams via standing waves. This flow cell will be constructed such that reversible flow will be possible and i will allow undiluted collection of the sample for re-analysis. Second, the flow cell will be coupld to an optimized optical system that uses a high-speed sCMOS camera for collection and a laser line that perpendicularly crosses the flow streams. The camera can collect 2048 x 8 pixels frames at rates of 25,655 fps, which will allow high speed collection of all flow streams using a single sensor. Importantly, we have developed a filter based optical dispersion system that allows collection of multiple colors within the same frame of the sensor. This will allow us to create a single detector instrument that can collect 3 spectral regions, thus reducing instrument cost and complexity. The third task will create a high-speed data acquisition that strips the data from the 2048 pixel x 8 pixel 16-bit data depth frames and converts it into standard flow cytometry file format. This will allow our instrument to be immediately implemented within the data analysis pipeline that exists in most labs worldwide. Fourth, the three subsystems will be integrated into a single flow cytometer that can sample at 50 mL/min, support particle analysis rates as high as 100,000 per second, collect three spectral regions of emitted light for fluorescence or scatter measurement, and provide sensitivities as low as few thousand fluorophores per particle. This cytometer will be engineered to be commercially robust and support real world users. Importantly, it will be constructed to be available at an average selling price of $100K per instrument. Finally, we will demonstrate the detection of mammalian and bacterial cells spiked in liquid samples at concentrations as low as 1 cell/L. Initial efforts will simply spike cells into clear buffers, but subsequent work will spike cells into clarified or lysed natural samples. This will be done to effectively mimic anticipated applications. Success in developing the HVPAfc will create an instrument that is immediately valuable for high volume applications and provide the basis for future flow cytometers that can address a myriad of additional application areas.