Department of Haematology and Transfusion Medicine, Paul Ehrlich Institute, Langen, Germany.
Haemophilia. 2014 Mar;20(2):e164-70. doi: 10.1111/hae.12333. Epub 2013 Nov 29.
Under certain circumstances, the determination of coagulation factor VIII (FVIII) is hampered by assay discrepancies between clotting and chromogenic approaches. These are observed in certain patients' plasma as well as in certain concentrates. We intended to develop a novel assay for the quantification of coagulation FVIII which reflects the physiological situation better than the established assays. It is based on plasma without chelation of divalent cations and simultaneously minimizes the generation of activated factors which could function as uncontrolled triggers of coagulation. FVIII deficient plasma is prepared with the aid of biotinylated antibodies against FVIII from normal plasma in presence of inhibitors of contact activation. To start the assay only tiny amounts of activated FIX serve as trigger. The FVIII determination is performed in a kinetic experiment and is based on the cleavage of a fluorogenic substrate for activated FX. FVIII concentrations between 0.01 and 1 IU mL(-1) are easily determined. Plasma-derived and recombinant FVIII concentrates were compared. All plasma-derived concentrates were found to contain FVIII activities within the specification of the manufacturer. Recombinant concentrates yielded only 35-50% of the claimed potency. The novel in vivo-like assay avoids the undue advantage or disadvantage of certain product characteristics by eliminating unphysiological assay conditions. Its usefulness could turn out in future experiments with plasma from haemophilia A patients.
在某些情况下，凝血因子 VIII（FVIII）的测定受到凝固和显色方法之间的检测差异的阻碍。这些在某些患者的血浆以及某些浓缩物中都有观察到。我们旨在开发一种新的凝血因子 VIII 定量检测方法，该方法比已建立的检测方法更能反映生理情况。它基于未螯合二价阳离子的血浆，同时最大限度地减少了激活因子的产生，这些激活因子可能作为不受控制的凝血触发因素发挥作用。使用来自正常血浆的针对 FVIII 的生物素化抗体，在接触激活抑制剂的存在下，制备 FVIII 缺乏的血浆。仅需少量激活的FIX 即可启动检测。FVIII 测定在动力学实验中进行，基于对激活 FX 的荧光底物的裂解。可以轻松测定 0.01 至 1IU mL(-1) 之间的 FVIII 浓度。比较了血浆衍生的和重组的 FVIII 浓缩物。所有血浆衍生的浓缩物均被发现含有制造商规定范围内的 FVIII 活性。重组浓缩物仅产生声称效价的 35-50%。新型的体内样检测通过消除非生理的检测条件，避免了某些产品特性的不当优势或劣势。在未来对血友病 A 患者血浆的实验中，它可能会变得有用。
