Thrombin generation in plasma of healthy adults and children: Chromogenic versus fluorogenic thrombogram analysis

Summary

Coagulation tests and coagulation factor assays have been complemented recently with experimental tests to measure the total amount of thrombin formed.We have presently analyzed thrombin generation of healthy adult and paediatric plasma samples via a fluorogenic and a chromogenic method.The chromogenic method was performed on the fully automated Behring Coagulation System^® (BCS^®) and fluorogenic assays via Calibrated Automated Thrombography ^® (CAT),after coagulation induction by various tissue factor (TF) concentrations.Sample distribution and variability were analyzed for the four main coagulation parameters, derived via computerized curve analysis in each method. Results for both methods were correlated.At the recommended TF concentration (300 pM), thrombin generation via BCS was less variable than via CAT (1–6 pM), but at comparable TF concentrations (1–6 pM), the CAT sensitivity was higher than that of BCS. Inhibition of intrinsic coagulation with the anti-factor VIII antibody BO2C11 revealed that the BCS detected extrinsic coagulation exclusively, at all TF concentrations tested. In contrast, at low TF concentrations (1 and 2.5 pM), via CAT, intrinsic coagulation pathway amplification was measured. At standardizedTF concentrations (300 pM in BCS vs. 2.5 pM in CAT), different reference values between adults and children were found, for all parameters, except Tmax. In adult samples, the best correlation between both methods was observed for ETP_CAT versus ETP_BCS and for Peak height_CAT versus Cmax_BCS, when thrombin generation was exclusively extrinsic (300 pM in BCS vs. 6 pM in CAT). In conclusion, differential thrombin generation characteristics in BCS and CAT are relevant for their clinical applicability.

Keywords

Tissue factor - coagulation assays - endogenous thrombin potential - fibrinogen

• References

• 1 Baglin T. The measurement and application of thrombin generation. Br J Haematol 2005; 130: 653-661.
• 2 Hemker HC, Béguin S. Phenotyping the clotting system. Thromb Haemost 2000; 84: 747-751.
• 3 Brummel-Ziedins KE, Pouliot RL, Mann KG. Thrombin generation: phenotypic quantitation. J Thromb Haemost 2003; 2: 281-288.
• 4 Hemker HC, Dieri RA, De Smedt E. et al. Thrombin generation, a function test of the haemostaticthrombotic system. Thromb Haemost 2006; 96: 553-561.
• 5 Biggs R, Macfarlane RG. Human blood coagulation and its disorders. Oxford Blackwell: 1953. .
• 6 Macfarlane RG, Biggs R. A thrombin generation test. J Clin Pathol 1953; 6: 3-7.
• 7 Hemker HC, Willems GM, Béguin S. A computer assisted method to obtain the prothrombin activation velocity in whole plasma in whole plasma independent of thrombin decay processes. Thromb Haemost 1986; 56: 9-17.
• 8 Hemker HC, Wielders S, Kessels H. et al. Continuous registration of thrombin generation in plasma, its use for the determination of the thrombin potential. Thromb Haemost 1993; 71: 617-624.
• 9 Hemker HC, Giesen P, Al Dieri R. et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003; 33: 4-15.
• 10 Jacquemin MG, Desqueper BG, Benhida A. et al. Mechanism and kinetics of factor VIII inactivation: study with an IgG4 monoclonal antibody derived from a hemophilia A patient with inhibitor. Blood 1998; 92: 496-506.
• 11 Josso F, Prou-Wartelle O. Interaction of tissue factor and factor VII at the earliest phase of coagulation. Thromb Diath Haemorrh 1965; 17: 35-44.
• 12 Luddington R, Baglin T. Clinical measurement of thrombin generation by calibrated automated thrombography requires contact factor inhibition. J Thromb Haemost 2004; 2: 1954-1959.
• 13 Baglin T. The measurement and application of thrombin generation. Br J Haematol 2005; 130: 653-661.
• 14 Mannhalter C, Quehenberger P. Laboratory methods in the haemostatic laboratory. Thromb Haemost 2006; 96: 545-546.
• 15 Gerotziafas GT, Depasse F, Busson J. et al. Towards a standardization of thrombin generation assessment: The influence of tissue factor, platelets and phospholipids concentration on the normal values of Thrombogram-Thrombinoscope assay. Thromb J 2005; 3: 16.
• 16 Al Dieri R, Alban S, Béguin S. et al. Thrombin generation for the control of heparin treatment, comparison with the activated thromboplastin time. J Thromb Haemost 2004; 2: 1003-1007.
• 17 Lawrie AS, Gray E, Leeming D. et al. A multicentre assessment of the endogenous thrombin potential using a continuous monitoring amidolytic technique. Br J Haematol 2003; 123: 335-341.
• 18 Haidl H, Cimenti C, Leschnik B. et al. Age-dependency of thrombin generation measured by means of calibrated automated thrombography (CAT). Thromb Haemost 2006; 95: 772-775.
• 19 Andrew M, Vegh P, Johnston M. et al. Maturation of the hemostatic system during childhood. Blood 1992; 80: 1998-2005.