Mortality associated with congenital heart disease (CHD) accounts for up to 50% of pediatric deaths with over 48% of CHD mortality occurring in children less than 1 year of age. Post-operative infections contribute significantly to this mortalty with a recent study indicating a mortality rate of nearly 25% associated with post- operative infections. Nearly all children with CHD require surgical correction of their lesions necessitating the use of cardiopulmonary bypass (CPB). However, CPB stimulates the immune system to produce pro- inflammatory cytokines that are counteracted by a compensatory anti-inflammatory response generated by the immune system. In some children, this compensatory anti-inflammatory response remains prolonged, leading to an immunoparalyzed state in which the pediatric patients are no longer able to fend off pathogens resulting in post-operative infections. Importantly, immunoparalysis can be revered in critically ill children by treating them with granulocyte macrophage colony-stimulating factor (GM-CSF). Even though immunoparalysis is associated with the dysregulation of immune function and the devastating consequences of post-operative infections, it remains a challenge for clinicians to identify those children who are immunoparalyzed and may benefit from GM-CSF to prevent post-operative infections. One major reason for the limited identification of children with immunoparalysis is the lack of validated immunomonitoring platforms that can allow real-time determinations of immunoparalysis. Two current methods exist to detect immunoparalysis: whole blood stimulation assay, which involves measurements of the capacity of peripheral blood leukocytes to produce inflammatory cytokines following pathogen stimulation using ELISA, and flow cytometry for enumeration of peripheral blood monocytes expressing HLA-DR receptors. The whole blood stimulation assay is time-consuming and can only provide the clinician with a determination of immunoparalysis in 72 hrs at the earliest. Flow cytometry for enumeration of peripheral blood monocytes expressing HLA-DR receptors can provide fast results; however it does not reflect the functional status of monocytes. Further, current protocols for isolation and functional testing of key leukocyte subpopulations (such as monocytes) require blood volumes that make such testing dangerous in small children. Thus, to understand the impact of immunoparalysis on outcomes for children following CPB as well as to identify functional status of key leukocyte subpopulations that are dysregulated in children who are immunoparalyzed, there exists a significant need for a real-time, functional immunophenotyping method that can achieve accurate, multiplexed functional cellular characterization of subpopulations of immune cells using only minute amounts of patient blood. To address this need, our central objective of this research is to develop an integrated microfluidic immunosensing platform for efficient isolation, enrichment, enumeration, and sensitive multiplexed functional immunophenotyping of subpopulations of immune cells from blood specimens. To extend our research to make clinical impact, we propose to implement our microfluidic immunomonitoring technology to further determine the incidence and impact of immunosuppression in critically ill children and its functional connection to post-operative infections. The Specific Aims of this collaborative R01 research thus are: (Aim 1) To develop and validate an integrated microfluidic immunophenotyping assay (MIPA) device for rapid, accurate, and sensitive parallel detection of multiple cytokines secreted by immune cells; (Aim 2) To develop and validate an integrated MIPA device for efficient isolation, enrichment, enumeration, and multiplexed functional immunophenotyping of subpopulations of immune cells from blood specimens; (Aim 3) To utilize the MIPA device to identify immunoparalyzed pediatric patients following CPB and determine its predictive power for post-operative infections.