Fig. 2

IFE of AMC fluorophore and its correction via calibration or normalization. FVIII-DP was supplemented with 1 IU/mL FVIII to normalize hemophilia plasma, or not, and was subsequently premixed with the indicated concentrations of AMC prior to initiating coagulation with Ca²⁺ and substrate. Raw fluorescent data were produced by the CAT microplate reader and software and analyzed in several different ways: (A, B) raw AMC fluorescence in relative fluorescent units (RFU), (C, D) internally calibrated TG curves via a thrombin calibration coefficient (see Materials and Methods), (E, F) normalized-uncalibrated curves, (G, H) calibrated TG curves (via TS software), (I, J) calibrated TG curves (via OR software), and (L, M) calibrated TG curves (via SH software). Uncalibrated curve data were produced by differentiating the AMC curves observed in (A, B). Calibrated curves were produced using TS software, our in-house OR software, which uses published algorithms similar to CAT calibration, or SH software, our second in-house app based on CAT algorithm. An asterisk (*) next to the indicated concentrations in panels G & H denotes high AMC concentrations in which commercial TS software did not report TG curves, possibly due to their noisy appearance as suggested by the TG curves reported by OR and SH software apps at these high concentrations. Normalized-uncalibrated curves were produced by normalizing each uncalibrated curve pairing of hemophilic sample and normalized hemophilia sample (hemophilic plasma supplemented with FVIII) at each pre-spiked AMC concentration against the TPH value of the normalized plasma sample in each pairing. TG was recorded for 40–60 min. Assay conditions: 63 µL of FVIII-DP, 1 µL of FVIII (1 IU/mL), 16 µL of AMC at indicated concentrations, 20 µL of PPP trigger, and 20 µL of FluCa.