Calibrated Automated Thrombin Generation Measurement in Clotting Plasma

TLDR

Calibrated automated thrombography quantifies thrombin activity in clotting plasma, with or without platelets, by monitoring fluorogenic substrate cleavage and comparing it to a reference thrombin standard in a parallel non‑clotting sample. The assay compensates for the non‑linear reaction rate by using a reference thrombin standard, eliminates the need for excess substrate, and employs standardized conditions that balance experimental variation with sensitivity to pathological changes. The assay shows low analytical variability (≈3 % within‑run, <5 % intra‑individual PPP, <8 % PRP, 15–19 % interindividual) and can detect all clotting factor deficiencies except factor XIII, the effects of anticoagulants, platelet inhibitors, von Willebrand disease, and APC system disorders.

Abstract

Calibrated automated thrombography displays the concentration of thrombin in clotting plasma with or without platelets (platelet-rich plasma/platelet-poor plasma, PRP/PPP) in up to 48 samples by monitoring the splitting of a fluorogenic substrate and comparing it to a constant known thrombin activity in a parallel, non-clotting sample. Thus, the non-linearity of the reaction rate with thrombin concentration is compensated for, and adding an excess of substrate can be avoided. Standard conditions were established at which acceptable experimental variation accompanies sensitivity to pathological changes. The coefficients of variation of the surface under the curve (endogenous thrombin potential) are: within experiment ∼3%; intra-individual: &lt;5% in PPP, &lt;8% in PRP; interindividual 15% in PPP and 19% in PRP. In PPP, calibrated automated thrombography shows all clotting factor deficiencies (except factor XIII) and the effect of all anticoagulants [AVK, heparin(-likes), direct inhibitors]. In PRP, it is diminished in von Willebrand’s disease, but it also shows the effect of platelet inhibitors (e.g. aspirin and abciximab). Addition of activated protein C (APC) or thrombomodulin inhibits thrombin generation and reflects disorders of the APC system (congenital and acquired resistance, deficiencies and lupus antibodies) independent of concomitant inhibition of the procoagulant pathway as for example by anticoagulants.