Abstract
BackgroundThrombin generation assay was developed several years ago to mimic physiological coagulation mechanisms but it had important limitations. Thrombogram-Thrombinoscope assay using a fluorogenic substrate, allows obtaining thrombin generation curves in non-defibrinated platelet rich plasma (PRP) in a fully automated manner.MethodsWe standardised the methodology of Thrombogram-Thrombinoscope and we evaluated the precision of thrombin generation parameters (lag-time, maximum concentration of thrombin [Cmax], time required to reach Cmax [Tmax] and endogenous thrombin potential ETP) using different concentrations of recombinant human tissue factor, platelets or phospholipids. Normal values of thrombin generation assay were established in optimal experimental conditions.ResultsIn the presence of low TF concentrations (final dilution of thromboplastin in plasma: 1/1000–1/2000) the Thrombogram assay showed intra-assay and inter-assay coefficients of variation lower than 9%. Thrombin generation parameters showed an important inter-individual variability and the coefficients of variation ranged from 18% to 50%. In PRP the lag-time, Cmax and Tmax but not the ETP, were influenced by TF concentration. Thrombin generation parameters were not influenced by variations of platelet concentration from 50 × 109/l to 400 × 109/l. The addition of synthetic procoagulant phospholipids in PPP strongly influenced all the parameters of thrombogram. For all the parameters of thrombogram a threshold effect was observed in the presence of phspholipid concentrations equal or higher to 4 μM. In frozen-thawed PRP the lag-time and the Tmax were significantly reduced and the Cmax was increased compared to the fresh PRP, but the ETP, the intra assay and the inter-assay coefficients of variation were similar in both test-systems.ConclusionThrombogram-Thrombinoscope assay performed in fresh or in frozen-thawed PRP has an acceptable precision, with low inter-assay and intra-assay coefficient of variations. The concentration of TF is determinant for the normal values of the studied parameters of thrombin generation. When the assay is performed in PPP, thrombin generation parameters are influenced by the concentration of procoagulant synthetic phospholipids. The optimal experimental conditions were obtained in the presence of 1/1000 final dilution of thromboplastin, a platelet count higher than 50 × 109/l and a synthetic phospholipid concentration higher than 4 μM.
Highlights
Thrombin generation assay was developed several years ago to mimic physiological coagulation mechanisms but it had important limitations
The most sophisticated version of thrombin generation assay using a chromogenic substrate developed by Hemker's group was less time-demanding than the previous one, since it was fully automated accompanied with a software for the calculation of the area under thrombin generation curve [13]
We examined the intra-assay, the inter-assay and the inter-individual coefficients of variations of thrombin generation lag-time, the maximum concentration of thrombin (Cmax), the time required to achieve maximum thrombin generation (Tmax) and the endogenous thrombin potential (ETP) in fresh platelet rich plasma (PRP) and in frozen-thawed PRP after triggering tissue factor (TF) pathway in plasma from healthy volunteers and we established the normal values for each parameter of the thrombogram
Summary
Thrombin generation assay was developed several years ago to mimic physiological coagulation mechanisms but it had important limitations. The global clotting times are mandatory for the detection of clotting factor deficiencies and the biological monitoring of treatment with coumarins and unfractionated heparin but they have a very limited value in the detection of hypercoagulable states [1,2]. They are not significantly influenced by the presence of therapeutic concentrations of LMWHs or the indirect FXa inhibitors such as fondaparinux [3]. At least two important components of blood coagulation (i.e. platelets and fibrinogen/fibrin) were absent [14]
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