(598d) Multiplexed Cytokine Analysis by Microengraving for Characterizing Human Immune Responses

The large diversity of cells that comprise the human immune system requires methods that can resolve the individual contributions from specific subsets to an immunological response. Correlating the lineages of individual cells with their functions (cytokine release, cytotoxicity) will improve the understanding of human immunology, and provide insights to the mechanisms of immune diseases., Two additional considerations for practical application in clinical studies are that methods should be amenable for characterizing small numbers of cells (10^4-10^6) and sufficient to detect target cells at low frequencies (1 in 10^4 to 10^6). This talk will describe a single-cell technology for high-throughput and multiplexed detection of individual immune cells. Through loading cells in an array of microwells, more than 80,000 cells are examined and four cytokines are measured simultaneously from each cell in a single process called microengraving. Together with image-based cytometry, an immunological profile consisting of both phenotype and functions for each individual cell is generated. In order to characterize the mass transfer and analytes binding process during microengraving, we constructed a mathematical model for this system and calculated the concentration of cytokines at different time points. Results of simulations indicate a linear range of surface binding that allows the quantification of the rates of secretion of cytokines from single cells. The application of this high-throughput, high-content single-cell technology to challenges in diagnosing allergies and characterizing autoreactive T cells in multiple sclerosis will be presented.