Optimization of plasma fluorogenic thrombin-generation assays

We optimized fluorogenic thrombin-generation assays with regard to sample volume, calibration, analytic corrections, and activation reagents. Lower sample volumes (40 vs 80 μL) were associated with better recovery of thrombin activity, lower interference due to absorbance of light, and higher total thrombin generation (area under the curve), even using internal standards to calibrate plasma samples. With lower sample volumes, there was no advantage to internal calibration of samples without obvious interference (hemolysis). Previously developed corrections for measured vs expected fluorescence units, residual thrombin-α₂-macroglobulin activity, and hemolysis improved the analytic accuracy of the assay. An optimized assay with a 40-μL sample volume, analytic corrections, and a corn trypsin inhibitor to block contact activation showed that 0.6 pmol/L tissue factor activator was better than 5 pmol/L at differentiating healthy subjects from patients with sepsis while demonstrating good reproducibility (area under the curve, 4% within-run and 7% between-run coefficient of variation).