Fig. 6

Effect of CAT calibration on TG curves in procoagulant plasma samples. Antithrombin deficient plasma (ATIII-DP) was treated with or without heparin and the indicated TF concentration to assess the effect of calibration via different software apps in procoagulant samples. Raw data was produced by the CAT assay 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 CBER algorithm), and (I, J) calibrated TG curves (via SH software). Uncalibrated curve data were produced by differentiating the AMC curves observed in (A, B). CAT calibrated curves were produced by our in-house OR and SH software apps. 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 60 min. Assay conditions: ATIII-DP with 0.2 U/mL of normal pooled plasma with or without heparin (0.03 USP/mL), TF (0.12–20 pM), tPA (0.13 µg/mL), thrombomodulin (12.5 nM), PC:PS vesicles (4 µM) and custom FluCa mixture (800 µM ZGGR-AMC and calcium chloride)