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

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 cells per second and sample delivery rates of 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 x pixels frames at rates of 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 spectral regions thus reducing instrument cost and complexity The third task will create a high speed data acquisition that strips the data from the pixel x pixel 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 mL min support particle analysis rates as high as 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 $ K per instrument Finally we will demonstrate the detection of mammalian and bacterial cells spiked in liquid samples at concentrations as low as 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 PUBLIC HEALTH RELEVANCE The proposed project will develop a high volume parallel acoustic flow cytometer for analysis of large volume samples This instrument will have immediate value for many applications such as the detection of bladder cancer cells in urine pathogenic bacteria in milk or water or rare cells in lysed blood samples and thus will have a large impact on the field and the marketplace The proposed research will have significant impact in the detection and diagnosis of many diseases and pathogens of interest to human health