Enhanced factor VIII activity measurements using ROTEG and factor VIII–/– mice whole blood

Abstract

To cite this article: Landskroner KA, Olson NC, Jesmok GJ. Enhanced factor VIII activity measurements using ROTEG and factor VIII-/-mice whole blood. J Thromb Haemost 2004; 2: 2274-5.To quantify the rate of fibrin formation after recombinant FVIII (rFVIII) administration to FVIII-deficient mouse whole blood we have employed the rotational thromboelestography (ROTEG, Pentapharm) assay system. This system demonstrated greater sensitivity than traditional assay systems and allows the analysis of clot (fibrin) formation in real time. This system uses FVIII-deficient mouse whole blood and provides a more relevant fibrin formation assay system than plasma-based systems. In contrast to ROTEG, current assays used to measure FVIIIa-dependent fibrin clot formation, such as the activated partial thromboplastin time (APTT), are less sensitive. Reasons for the decreased sensitivity are due, in part, to the absence of platelets that are necessary for optimal tenase and prothrombinase assembly. While there has always been interest in measuring low FVIIIa activity, i.e. < 0.001 U mL , the technology and methodology were not available. Thromboelastography (TEG) systems have been available for decades; however, recent ROTEG technology has improved ease of use and reproducibility [1,2]. In order to evaluate the detection sensitivity of ROTEG at low levels of FVIII we examined the thromboelastographic profile of FVIII-/-mice [3] whole blood supplemented with rFVIII over a 100 000-fold concentration range. The ability to generate a clear doseresponse relationship between FVIII levels of 0.00001 U mL )1 (0.001%) to 1 U mL )1 (100%) and the rate of fibrin formation demonstrates the enhanced sensitivity of the approach. Briefly, blood from mice was drawn from the descending vena cava under anesthesia and immediately anticoagulated with 3.8% citrate in a 1 : 9 dilution (citrate : blood). For comparison, blood from C57BL/6 mice (FVIII+/+, background strain) was used as control. Blood samples were pooled from three to five animals for analysis. Within 8 h of sampling the blood was assayed with the ROTEG system using the NATEG protocol provided by the manufacturer [1,2]. Coagulation was allowed to proceed without the exogenous addition of tissue factor. Recombinant FVIII (Refacto, Wyeth) at each dilution was added in a total volume of 3.3 lL to 330 lL of whole blood. The coagulation reaction was started by the addition of 20 lL of 200 mM CaCl 2 . Using this protocol the following measurements were made: clotting time (CT), the time delay from start of sample run to point where trace is 1 mm wide; clot formation time (CFT), the time between the 1 mm wide point to the 20 mm wide point; and alpha angle, which is an indirect measurement of the rate of clot formation [1,2]. The tests were halted after the alpha angle was calculated by the software program, or 2 h, whichever happened first. Experiments consisted of drawing blood from three to five animals, pooling the blood, and adding dilutions of rFVIII. Each experiment was repeated three to f...

Coagulation phenotype of wild-type mice on different genetic backgrounds

“…TEG is a wellaccepted method to test patients' coagulation status and has been applied to a variety of preclinical haemophilia models including mice to assess the efficacy of administered clotting factors. 7,8 Activated partial thromboplastin time (aPTT), prothrombin time (PT) and fibrinogen levels are also indicators of the influence of haemophilia drugs on the coagulation system 9,10 and are frequently included in the evaluation of preclinical efficacy and safety of experimental therapies.…”

Thromboelastography measurements of whole blood from factor VIII‐deficient mice supplemented with rFVIII

“…Pooled blood was used to diminish assay variability and several pools were used to acquire data over entire dose‐relationship. Reprinted with permission [23].…”

Preclinical safety and efficacy of a new recombinant FIX drug product for treatment of hemophilia B

“…The efficacy of BAX326, defined as statistically significantly reduced blood loss compared with buffer, was tested in three mouse models of primary pharmacodynamics (FIX ko mice: B6.129P2-F9 tm1Dws ). A carotid occlusion model (COM), and thrombelastography (TEG), which has been used in a variety of preclinical models of hemophilia [9,10], were used to compare the efficacy of BAX326 with that of the previously licensed FIX products. Animals received intravenous (iv; clinical application route) prophylactic treatment with 75 IU/kg of BAX326 or licensed rFIX, and, in the carotid occlusion model, licensed pdFIX.…”