ACT-LR whole-blood clotting assay quantifies hFVIII levels by clotting time in hemophilia A mouse blood. A standard curve was generated by collecting hemophilia A mouse blood and adding recombinant hFVIII at 6 different known concentrations: 0.5%, 1.2%, 3.3%, 13.3%, 33.3%, and 100% normal human levels. A clean blood sample (free of significant tissue factor) was used to generate the standard curve; sample integrity was verified by blood that would not clot for more than 400 seconds (assay upper limit) without addition of hFVIII. The log of the clotting time (seconds) has a linear relationship with the amount of hFVIII in the blood (R² = 0.97; error ± SD).