dc.contributor.author | Hu, Wenting | |
dc.contributor.author | Soper, Steven A. | |
dc.contributor.author | Jackson, J. Matt | |
dc.date.accessioned | 2019-11-07T22:05:05Z | |
dc.date.available | 2019-11-07T22:05:05Z | |
dc.date.issued | 2019-02-28 | |
dc.identifier.citation | Hu, 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.9b00021 | en_US |
dc.identifier.uri | http://hdl.handle.net/1808/29724 | |
dc.description | This 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.abstract | We 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.publisher | American Chemical Society | en_US |
dc.rights | Copyright © 2019 American Chemical Society | en_US |
dc.title | Time-Delayed Integration–Spectral Flow Cytometer (TDI-SFC) for Low-Abundance-Cell Immunophenotyping | en_US |
dc.type | Article | en_US |
kusw.kuauthor | Hu, Wenting | |
kusw.kuauthor | Soper, Steven A. | |
kusw.kuauthor | Jackson, J. Matt | |
kusw.kudepartment | Chemistry | en_US |
dc.identifier.doi | 10.1021/acs.analchem.9b00021 | en_US |
dc.identifier.orcid | https://orcid.org/0000-0002-8292-7058 | en_US |
kusw.oaversion | Scholarly/refereed, author accepted manuscript | en_US |
kusw.oapolicy | This item meets KU Open Access policy criteria. | en_US |
dc.identifier.pmid | PMC6554645 | en_US |
dc.rights.accessrights | OpenAccess | en_US |