PROJECT SUMMARY In this proposal we build on our relevant published work1-14, and ongoing research activities supported by an active NHLBI R01 (R01HL133574, PI: Gurkan, Little), titled: “Standardized Monitoring of Cellular Adhesion to Improve Clinical Care in Sickle Cell Disease”, an active FDA R01 (R01FD005341, PI: Manwani), titled: “Phase 2 Trial of Gamunex (Intravenous Gammaglobulin) for Sickle Cell Acute Pain”, and an active NSF Award (1552782, PI Gurkan): “CAREER: Biomechanics of Red Blood Cell Adhesion and Deformability”. We have developed red blood cell (RBC) and white blood cell (WBC) biomarker assays for more robust phenotypic analyses in sickle cell disease (SCD)1-14, supported by an NHLBI R01 (R01HL133574) and an FDA R01 (R01FD005341) award, as described above. In this project we build on our expertise in novel SCD biomarker assay development to uniquely address the following high priority areas identified in OTA- 19-007 for Cure Sickle Cell Initiative: (1) Develop and validate assays to quantitatively determine cellular distribution and semi-quantitative cellular expression of fetal, adult and/or sickle hemoglobin. (2) Develop and validate assays to assess red blood cell rheology, including microfluidics and imaging flow cytometry sickling assays. In this project we propose advanced analytical and clinical validation of integrated clinical microfluidic assays to assess RBC adhesion, deformability, cellular hemoglobin distribution, cellular density (as mass to volume ratio for each individual cell), and whole blood rheology as candidate biomarkers to assess response to curative therapies in SCD.