Summary/Abstract Hemophilia A results from a deficiency of FVIII-activity and can result in severe bleeding. Head bleeds can be debilitating and joint-bleeds require major medical intervention. In this Phase IIB (PIIB) effort, we will leverage our CE-marked FVIII fluorogenic substrate assay (FSA) to develop a cost-effective point-of- care (POC) solution for measuring FVIII and emicizumab with a 15-minute turnaround time. Hemophilia A is an X-linked recessive genetic disorder with an incidence of 1 in 5000 live male births. For patients with hemophilia A, lifelong treatment is required to provide adequate and therapeutic FVIII or emicizumab FVIII-like levels. FVIII replacement monitoring is essential to maintaining optimal dosing. Acute bleeding and surgeries require knowledge of FVIII and/or FVIII-like levels for proper management. There are currently no approaches at the point-of-care for FVIII or FVIII-like measurements, resulting in challenges to implementation and access. We have developed three innovations: (1) a high-sensitivity CE-marked FVIII fluorogenic assay with bovine reagents and an emicizumab assay with human reagents, (2) pilot production test cartridges that employ 4D microfluidics, a novel innovation we have developed for full sample-to- answer processing, and (3) a pilot production hemophilia monitoring device and app that meets all CE- marking and FDA 510k design control requirements. Three aims are outlined to attain data for CE-marking and FDA 510k clearance: (1) Assess the accuracy and precision of all steps in our CLIA-waivable 4D sample- to-answer microfluidic cartridge, consumables, and controls. (2) Characterize hemophilia monitoring solution for turnaround time (TAT), analytical precision, interferences, ranges, limits of detection, and blind sample analysis for FVIII and FVIII-like activity. (3) Perform clinical validation with Boston Children’s Hospital (N = 300) and a contract research organization (CRO) (N = 1200). In all these studies, we will continue to work with the World Federation of Hemophilia (WFH) to ensure we have an approach for all patients worldwide. The success of developing a hemophilia POC diagnostic will allow a precision medicine approach for the management of hemophilia A. Home use will allow for more frequent measurement of FVIII levels to ensure therapeutic levels prior to engaging in physical activity and to assess correct dosage administration, including potential missed doses. The approach can be utilized to create FVIII activity trends which can be translated into immediate pK data, which can alert care providers to test for inhibitors. It will allow newer therapies, including emicizumab, extended half-life PEGylated FVIII, and gene therapies to be monitored. The results of our efforts will be a human-centered, hemophilia monitoring device and app that will provide accurate and easy-to-use tests with the goals of decreasing risk of unexpected bleeding and increasing patient care in all se...