Key result
Optimized fluorogenic thrombin-generation assay improves analytic accuracy and better differentiates sepsis patients from healthy subjects.
Why the study?
Optimization of fluorogenic thrombin-generation assays was needed to improve analytic accuracy and differentiation between healthy subjects and patients with sepsis.
Optimization of fluorogenic thrombin-generation assays with lower sample volumes and specific analytic corrections improves accuracy and clinical differentiation.
May refine thrombin-generation testing in sepsis; leaves open prospective validation before clinical adoption.
We optimized fluorogenic thrombin-generation assays with regard to sample volume, calibration, analytic corrections, and activation reagents. Lower sample volumes (40 vs 80 microL) 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-alpha(2)-macroglobulin activity, and hemolysis improved the analytic accuracy of the assay. An optimized assay with a 40-microL 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).
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Chandler et al. (2009) studied Sepsis. Optimized assay conditions (40-microL sample volume, 0.6 pmol/L tissue factor activator) vs. 80-microL sample volume, 5 pmol/L tissue factor activator was evaluated on Thrombin generation (area under the curve) and differentiation of healthy subjects from patients with sepsis. An optimized fluorogenic thrombin-generation assay using a 40-microL sample volume and 0.6 pmol/L tissue factor activator improved analytic accuracy and differentiation of sepsis patients from healthy subjects.
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