Towards a standardization of thrombin generation assessment: The influence of tissue factor, platelets and phospholipids concentration on the normal values of Thrombogram-Thrombinoscope assay
Towards a standardization of thrombin generation assessment: The influence of tissue factor, platelets and phospholipids concentration on the normal values of Thrombogram-Thrombinoscope assay
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DOI：
10.1186/1477-9560-3-16
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发表时间：
2005-01-01
影响因子：
3.1
通讯作者：
Samama, Meyer M.
中科院分区：
文献类型：
--
作者：
Gerotziafas, Grigoris T.;Depasse, Francois;Samama, Meyer M.
背景：凝血酶生成试验是几年前发展起来的模拟生理凝血机制，但它有重要的局限性。使用荧光底物的Thrombogram-Thrombinoscope测定允许以全自动方式获得非脱钙富血小板血浆（PRP）中的凝血酶生成曲线。方法：标准化了血栓图-血栓镜的方法学，并对凝血酶生成参数的精密度进行了评价（滞后时间，凝血酶的最大浓度[Cmax]，达到Cmax所需的时间[Tmax]和内源性凝血酶潜力ETP）。结果：在低浓度TF（终浓度为1/1000-1/2000）条件下，凝血酶生成试验的批内和批间变异系数均小于9%。凝血酶生成参数显示出重要的个体间变异性，变异系数范围为18%至50%。在PRP中，TF浓度影响滞后时间、Cmax和Tmax，但不影响ETP。凝血酶生成参数不受血小板浓度从50 × 10（9）/l至400 × 10（9）/l变化的影响。在PPP中加入合成的促凝血磷脂强烈影响所有的血栓参数。对于所有参数的血栓阈值效应的存在下，观察到磷脂浓度等于或高于4 μ M。在冻融PRP的滞后时间和Tmax显着减少，Cmax增加相比，新鲜的PRP，但ETP，批内和批间变异系数是相似的，在两个测试system.Conclusion：Thrombogram-Thrombinoscope分析进行新鲜或在冻融PRP具有可接受的精密度，与低批间和批内变异系数。TF的浓度是凝血酶生成研究参数正常值的决定因素。当在PPP中进行测定时，凝血酶生成参数受促凝血合成磷脂浓度的影响。在凝血活酶终稀释度为1/1000、血小板计数高于50 × 10（9）/l和合成磷脂浓度高于4 μ M的条件下获得了最佳实验条件。
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 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 x 10(9)/l to 400 x 10(9)/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 mu M. 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.Conclusion: 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 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 x 10(9)/l and a synthetic phospholipid concentration higher than 4 mu M.