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dc.contributor.authorHu, Wenting
dc.contributor.authorSoper, Steven A.
dc.contributor.authorJackson, J. Matt
dc.date.accessioned2019-11-07T22:05:05Z
dc.date.available2019-11-07T22:05:05Z
dc.date.issued2019-02-28
dc.identifier.citationHu, W., Soper, S. A., & Jackson, J. M. (2019). Time-Delayed Integration-Spectral Flow Cytometer (TDI-SFC) for Low-Abundance-Cell Immunophenotyping. Analytical chemistry, 91(7), 4656–4664. doi:10.1021/acs.analchem.9b00021en_US
dc.identifier.urihttp://hdl.handle.net/1808/29724
dc.descriptionThis document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/acs.analchem.9b00021.en_US
dc.description.abstractWe describe a unique flow cytometer (TDI-SFC) for the immunophenotyping of low-abundance cells, particularly when cell counts are sample-limited and operationally difficult for analysis by fluorescence microscopy (>100 cells) or multiparameter flow cytometry (MFC, <10 000 cells). TDI-SFC combines the high spectral resolution of spectral flow cytometry (SFC) with a CCD operated in time-delayed integration (TDI) for improved duty cycle and sensitivity. Cells were focused with a 1D-sheathing microfluidic device, and fluorescence emission generated from a 488 nm laser was collected by epi-illumination and dispersed along one axis of a CCD by a spectrograph. Along the other axis, the CCD’s shift rate was clocked at a rate that closely matched the cells’ velocity through the field of view. This TDI-SFC format allowed the CCD shutter to remain open during signal acquisition, providing a duty cycle ∼100% and assurance that ∼95% cells were interrogated. We used fluorescent beads to optimize synchronization of TDI clocking with the sheathed-cell velocity and to improve sensitivity via the excitation intensity, epi-illumination numerical aperture, and integration time. TDI achieved integrated signals of 106 counts at a signal-to-noise ratio (SNR) of 610 for beads corresponding to a load of 4 × 105 antibodies. We also evaluated multiplexing capabilities by spectral deconvolution and undertook a proof-of-concept application to immunophenotype low-abundance cells; the demonstration consisted of immunophenotyping a model cell line, in this case SUP-B15 cells representing B-cell acute lymphoblastic leukemia (B-ALL). The B-ALL cell line was stained against a leukemic marker (terminal deoxynucleotidyl transferase, TdT), and we successfully used spectral unmixing to discriminate TdT(+) cells from TdT(−) cells even at low cell counts (∼100 cells). The TDI-SFC could potentially be used in any application requiring the immunophenotyping of low-abundance cells, such as in monitoring measurable residual disease in acute leukemias following affinity enrichment of circulating leukemia cells from peripheral blood.en_US
dc.publisherAmerican Chemical Societyen_US
dc.rightsCopyright © 2019 American Chemical Societyen_US
dc.titleTime-Delayed Integration–Spectral Flow Cytometer (TDI-SFC) for Low-Abundance-Cell Immunophenotypingen_US
dc.typeArticleen_US
kusw.kuauthorHu, Wenting
kusw.kuauthorSoper, Steven A.
kusw.kuauthorJackson, J. Matt
kusw.kudepartmentChemistryen_US
dc.identifier.doi10.1021/acs.analchem.9b00021en_US
dc.identifier.orcidhttps://orcid.org/0000-0002-8292-7058en_US
kusw.oaversionScholarly/refereed, author accepted manuscripten_US
kusw.oapolicyThis item meets KU Open Access policy criteria.en_US
dc.identifier.pmidPMC6554645en_US
dc.rights.accessrightsOpenAccessen_US


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