Towards a standardization of thrombin generation assessment: The influence of tissue factor, platelets and phospholipids concentration on the normal values of Thrombogram-Thrombinoscope assay

被引：142

作者：

Gerotziafas G.T. ^{[1
,2
]}

Depasse F. ^{[2
]}

Busson J. ^{[2
]}

Leflem L. ^{[2
]}

Elalamy I. ^{[1
]}

Samama M.M. ^{[1
,2
]}

机构：

[1] Service d'Hématologie Biologique, Hôpital Hôtel-Dieu de Paris, 75181 Paris Cedex 04

[2] LCL, Ivry sur Seine

来源：

Thrombosis Journal | / 3卷 / 1期

关键词：

Endogenous thrombin potential; Platelets; Thrombin; Thrombin generation; Thrombinoscope; Tissue factor;

D O I：

10.1186/1477-9560-3-16

中图分类号：

学科分类号：

摘要：

Background: Thrombin 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. Methods: We 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. Results: In 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 interindividual variability and the coefficients of variation ranged from 18% to 50%. In PRP the lagtime, Cmax and Tmax but not the ETP, were influenced by TF concentration. Thrombin generation parameters were not influenced by variations of platelet concentration from 50×109/1 to 400×109/1. 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 phospholipid 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. Conclusions: Thrombogram-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 PPP1 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 50×109/1 and a synthetic phospholipid concentration higher than 4 μM. © 2005 Gerotziafas et al., licensee BioMed Central Ltd.

引用

页数：29

共 30 条

[1]

Hemker H.C., Beguin S., Phenotyping the clotting system, Thromb. Haemost., 84, pp. 741-751, (2000)

[2]

Mann K.G., Butenas S., Brummel K., The dynamics of thrombin formation, Arterioscler. Thromb. Vasc. Biol., 23, pp. 17-25, (2003)

[3]

Gerotziafas G.T., Depasse F., Chakroun T., Van Dreden P., Samama M.M., Elalamy I., Comparison of the effect of fondaparinux and enoxaparin on thrombin generation during in vitro clotting of whole blood and platelet rich plasma, Blood Coag. Fibrinol., 15, pp. 149-156, (2004)

[4]

Bara L., Planes A., Samama M.M., Occurrence of thrombosis and haemorrhage, relationship with anti-Xa, anti-IIa activities, and D-dimer plasma levels in patients receiving a low molecular weight heparin, enoxaparin or tinzaparin, to prevent deep vein thrombosis after hip surgery, Br. J. Haematol., 104, pp. 230-240, (1999)

[5]

Mahe I., Drouet L., Chassany O., Grenard A.S., Caulin C., Bergmann J.F., Low molecular weight heparin for the prevention of deep venous thrombosis: A suitable monitoring in elderly patients?, Pathophysiol. Haemost. Thromb., 32, pp. 134-136, (2002)

[6]

Heizmann M., Baerlocher G.M., Steinmann F., Horber F.F., Wuillemin W., Anti-Xa activity in obese patients after double standard dose of nadroparin for prophylaxis, Thromb. Res., 106, pp. 179-181, (2002)

[7]

Provenzale L., Casolo P., Bifani I., Value of an original thrombogram in the early diagnosis of thrombosis and the recognition of potential thrombosis, Policlinico Chir., 59, pp. 257-279, (1952)

[8]

Macfarlane R.G., Biggs R., A thrombin generation test, J. Clin. Pathol., 6, pp. 3-7, (1953)

[9]

Pitney W.R., Dacie J., A simple method of studying the generation of thrombin in recalcified plasma, J. Clin. Pathol., 6, pp. 9-13, (1953)

[10]

Seegers W.H., A personal perspective on hemostasis and thrombosis (1937-1981), Semin. Thromb. Hemost., 7, pp. 177-207, (1981)

← 1 2 3 →