Multiplexed Single-Cell Leukocyte Enzymatic Secretion Profiling from Whole Blood Reveals Patient-Specific Immune Signature

Enzymatic secretion of immune cells (leukocytes) plays a dominant role in host immune responses to a myriad of biological triggers, including infections, cancers, and cardiovascular diseases. Current tools to probe these leukocytes inadequately profile these vital biomarkers; the need for sample preprocessing steps of cell lysis, labeling, washing, and pipetting inevitably triggers the cells, changes its basal state, and dilutes the individual cell secretion in bulk assays. Using a fully integrated system for multiplexed profiling of native immune single-cell enzyme secretion from 50 μL of undiluted blood, we eliminate sample handling. With a total analysis time of 60 min, the integrated platform performs six tasks of leukocyte extraction, cell washing, fluorescent enzyme substrate mixing, single-cell droplet making, droplet incubation, and real-time readout for leukocyte secretion profiling of neutrophil elastase, granzyme B, and metalloproteinase. We calibrated the device, optimized the protocols, and tested the leukocyte secretion of acute heart failure (AHF) patients at admission and predischarge. This paper highlights the presence of single-cell enzymatic immune phenotypes independent of CD marker labeling, which could potentially elucidate the innate immune response states. We found that patients recovering from AHF showed a corresponding reduction in immune-cell enzymatic secretion levels and donor-specific enzymatic signatures were observed, which suggests patient-to-patient heterogeneous immune response. This platform presents opportunities to elucidate the complexities of the immune response from a single drop of blood and bridge the current technological, biological, and medical gap in understanding immune response and biological triggers.