Fig. 4

Substrate consumption and its correction via calibration or normalization. FVIII-DP was supplemented with 1 IU/mL FVIII to normalize hemophilic plasma, or not, and was subsequently premixed with the indicated concentrations of substrate, ZGGR-AMC, prior to initiating coagulation with Ca²⁺ and substrate. Raw data was produced by the CAT microplate reader 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 curves data were produced by differentiating the AMC curves observed in (A, B). Calibrated curves were produced using TS, OR or SH apps all of which employed the same CAT correction algorithm. Normalized-uncalibrated curves were produced by normalizing each uncalibrated curve pairing of hemophilic and normalized 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 min. Assay conditions: 63 µL of FVIII-DP, 1 µL of FVIII (1 IU/mL), 16 µL of substrate ZGGR-AMC (at indicated concentrations), 20 µL of PPP trigger, and 20 µL of Ca²⁺ (calcium chloride buffer)