Fig. 1

Schematic of thrombin generation curve correction methods. This schematic is to describe the correction methods for thrombin generation (TG) and outline these different algorithms used in this study. (A) Fluorescence curves for plasma samples (green) and for calibrator wells (red) are recorded. (B) The first derivative of these fluorescence curves is taken to generate uncalibrated TG curves in relative fluorescence units (RFU) per minute (RFU/min), and (C) subsequently corrected in the indicated calibration methods: Internal Linear Calibration (1st derivate of the fluorescence curves, i.e., uncalibrated curves, are multiplied by the initial linear slope of the calibration curve; provides calibration without fluorescence artifact correction), TS Software (i.e., commercial CAT Thrombinoscope software; 1st derivate is multiplied by the thrombin calibration curve with thrombin-α₂ macroglobulin signal values subtracted), OR software (i.e., our in-house version of CAT coded in Origin LabTalk software), and SH software (a web-based interface written by one of us (CL) which performs calibrations and corrections in a similar way to the CAT software), see materials and Methods. A normalization method was also employed such that uncalibrated curves were normalized against the normal thrombin peak height (TPH) values measured in normalized plasma samples with added FVIII (i.e., FVIII-DP + 1 IU/mL FVIII).