A paper by members of CBM2’s Soper research group has just been published in Analytical Chemistry on February 28, 2019. The paper, by Wenting Hu, J. Matt Jackson, and Steven A. Soper, is titled “Time-Delayed Integration—Spectral Flow Cytometer (TDI-SFC) for Low-Abundance-Cell Immunophenotyping.”
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.