Time-Delayed Integration-Spectral Flow Cytometer (TDI-SFC) for Low-Abundance-Cell Immunophenotyping

Area of Science:

• Biotechnology
• Analytical Chemistry
• Cell Biology

Background:

• Analyzing low-abundance cells presents challenges for traditional fluorescence microscopy and multiparameter flow cytometry due to limited cell counts and operational difficulties.
• Existing methods struggle with sample-limited scenarios, hindering accurate immunophenotyping of rare cell populations.
• There is a need for sensitive and efficient techniques to analyze cells when sample quantity is a critical constraint.

Purpose of the Study:

• To introduce and characterize a unique time-delayed integration spectral flow cytometer (TDI-SFC) for improved immunophenotyping of low-abundance cells.
• To demonstrate the TDI-SFC's capability to enhance sensitivity and duty cycle for analyzing sample-limited cell populations.
• To validate the TDI-SFC's performance in a proof-of-concept application for detecting rare leukemia cells.

Main Methods:

• Developed TDI-SFC by integrating spectral flow cytometry (SFC) with a charge-coupled device (CCD) operated in time-delayed integration (TDI) mode.
• Utilized a 1D-sheathing microfluidic device for cell focusing and collected fluorescence emission dispersed by a spectrograph onto the CCD.
• Synchronized CCD clocking with cell velocity for near 100% duty cycle, optimizing signal acquisition and sensitivity using fluorescent beads for calibration.

Main Results:

• Optimized TDI-SFC achieved high signal-to-noise ratios (SNR) of 610 for fluorescent beads, demonstrating enhanced sensitivity.
• Successfully performed spectral deconvolution for multiplexing capabilities.
• Demonstrated proof-of-concept by immunophenotyping a low-abundance B-cell acute lymphoblastic leukemia (B-ALL) cell line (SUP-B15), discriminating TdT+ cells from TdT- cells even with ~100 cells.

Conclusions:

• The TDI-SFC system significantly enhances sensitivity and duty cycle for the immunophenotyping of low-abundance cells, overcoming limitations of conventional methods.
• This technology is suitable for analyzing sample-limited specimens, offering a powerful tool for rare cell detection.
• TDI-SFC holds potential for applications such as monitoring minimal residual disease in acute leukemias by analyzing enriched circulating leukemia cells.