Jha N K, Shestopal S A, Gourley M J, Woodle S A, Liang Y, Sarafanov A G, Weinstein M, Ovanesov M V
Office of Blood Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
Department of Physics, George Washington University, Washington, DC, USA.
Haemophilia. 2016 Sep;22(5):780-9. doi: 10.1111/hae.12943. Epub 2016 Apr 1.
The thrombin generation test (TGT) is used both as a global haemostasis assay, and to compare activities of coagulation factor concentrates that have been spiked into patient plasma. However, TGT has not been systematically optimized to evaluate factor VIII (FVIII) product potency.
To improve the sensitivity of TGT to FVIII and allow a comparative analysis of the thrombin generating capacities of FVIII concentrates against reference preparations with known FVIII activity.
Concentrations of TGT components (analytical variables) were assessed to maximize the linearity and range of responses to the concentration of FVIII.
We optimized the range and sensitivity of the TGT assay with respect to FVIII through the addition of FXIa to the assay. Other parameters that were adjusted, i.e. tissue factor (TF), procoagulant lipids and plasma concentrations, did not improve the ability of the assay to measure both high and very low levels of FVIII. In the optimized TF/FXIa-activated TGT assay, all thrombin generation curve parameters were suitable for FVIII quantification, but thrombin peak height and maximal velocity demonstrated better linearity in the desired FVIII range. We found that the optimized TF/FXIa-activated TGT has a wider range of sensitivity to FVIII than a commercially available TGT. Additionally, we demonstrated that the TF/FXIa-activated assay performs adequately by comparing potency measurements of five commercially available FVIII products using TGT and traditional chromogenic and one-stage clotting assays.
The optimized TGT assay can be used to quantify and compare the thrombin generating capacities of FVIII concentrates.
凝血酶生成试验（TGT）既用作整体止血测定，也用于比较添加到患者血浆中的凝血因子浓缩物的活性。然而，TGT尚未系统优化以评估凝血因子VIII（FVIII）产品的效价。
提高TGT对FVIII的敏感性，并对FVIII浓缩物与已知FVIII活性的参考制剂的凝血酶生成能力进行比较分析。
评估TGT组分的浓度（分析变量），以最大化对FVIII浓度的反应线性和范围。
我们通过在试验中添加FXIa来优化TGT测定对于FVIII的范围和敏感性。其他调整的参数，即组织因子（TF）、促凝脂质和血浆浓度，并未提高该测定法测量高和非常低水平FVIII的能力。在优化的TF/FXIa激活的TGT测定中，所有凝血酶生成曲线参数都适用于FVIII定量，但凝血酶峰高和最大速度在所需的FVIII范围内表现出更好的线性。我们发现，优化的TF/FXIa激活的TGT对FVIII的敏感性范围比市售TGT更宽。此外，我们通过使用TGT以及传统的发色和一步凝血测定法比较五种市售FVIII产品的效价测量，证明了TF/FXIa激活的测定法表现良好。
优化的TGT测定法可用于定量和比较FVIII浓缩物的凝血酶生成能力。
