Laser particle barcoding for multi-pass high-dimensional flow cytometry

Flow cytometry is a standard technology in life science and clinical laboratories used to characterize the phenotypes and functional status of cells, especially immune cells. Recent advances in immunology and immuno-oncology as well as drug and vaccine discovery have increased the demand to measure more parameters. However, the overlap of fluorophore emission spectra and one-time measurement nature of flow cytometry are major barriers to meeting the need. Here, we present multi-pass flow cytometry, in which cells are tracked and measured repeatedly through barcoding with infrared laser-emitting microparticles. We demonstrate the benefits of this approach on several pertinent assays with human peripheral blood mononuclear cells (PBMCs). First, we demonstrate unprecedented time-resolved flow characterization of T cells before and after stimulation. Second, we show 33-marker deep immunophenotyping of PBMCs, analyzing the same cells in 3 back-to-back cycles. This workflow allowed us to use only 10-13 fluorophores in each cycle, significantly reducing spectral spillover and simplifying panel design. Our results open a new avenue in multi-dimensional single-cell analysis based on optical barcoding of individual cells. ### Competing Interest Statement All authors have financial interests in LASE Innovation Inc., a company focused on commercializing technologies based on optical barcodes. The financial interests of N.M. and S.H.Y. were reviewed and are managed by Massachusetts General Brigham in accordance with their conflict-of-interest policies.