Illustration of kinetic bias leading to higher OS than CS activity of transgene-produced FVIII-SQ. Transgene-produced FVIII-SQ accelerates early FXa formation compared with native FVIII, resulting in a small increase in FXa concentration (picomolar range, pM). This leads to faster thrombin activation and clot formation in the OS assay, which may explain the higher OS measurements, given that this assay uses a kinetic end point (time to visible clot) within the first 1 to 2 minutes of the coagulation reaction. In contrast, the CS assay uses more dilute test samples and determines FXa concentrations after a longer, fixed incubation period of 5 minutes. At this point, FXa generation has been exponentially amplified (nanomolar range, nM) and presumably remains unaffected by the minute differences that occurred earlier in the reaction. The different timing of assay readout therefore leads to a kinetic bias between OS and CS assay, whereby the shorter assay (OS) reports higher FVIII activity values.