Optimization of the thrombin generation test components to measure potency of factor VIII concentrates.

INTRODUCTION: 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. AIMS: 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. METHODS: Concentrations of TGT components (analytical variables) were assessed to maximize the linearity and range of responses to the concentration of FVIII. RESULTS: 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. CONCLUSIONS: The optimized TGT assay can be used to quantify and compare the thrombin generating capacities of FVIII concentrates.

Predicting dosing advantages of factor VIIa variants with altered tissue factor-dependent and lipid-dependent activities.

BACKGROUND: Recombinant factor VIIa (rFVIIa) is an FX-cleaving coagulation enzyme licensed for the treatment of bleeding episodes in hemophiliacs with inhibitory antibodies. Even though the optimal dosing and comparative dose efficacy of rFVIIa remain poorly understood, genetic or chemical modifications of rFVIIa have been proposed, with the goal of achieving faster and longer hemostatic action. No ongoing trial is currently comparing rFVIIa variants with each other. OBJECTIVES AND METHODS: We used mathematical modeling to compare the pharmacokinetics, dose-response (pharmacodynamics) and dose-effect duration (pharmacokinetics/pharmacodynamics) of rFVIIa variants to predict their optimal doses. The pharmacodynamic (PD) model of FXa generation by FVIIa in complexes with tissue factor (TF) and procoagulant lipids (PLs) was validated against published ex vivo and in vitro thrombin generation (TG) experiments. To compare variants' safety profiles, the highest non-thrombogenic doses were estimated from the clinical evidence reported for the licensed rFVIIa product. RESULTS: The PD model correctly described the biphasic TF-dependent and PL-dependent dose response observed in TG experiments in vitro. The pharmacokinetic/PD simulations agreed with published ex vivo TG data for rFVIIa and the BAY 86-6150 variant, and explained the similar efficacies of a single dose of 270 µg kg(-1) (as reported in the literature) and repeated doses of 90 µg kg(-1) of unmodified rFVIIa. The duration of the simulated hemostatic effect after a single optimal dose was prolonged for rFVIIa variants with increased TF affinity or extended half-lives, but not for those with modulated PL activity. CONCLUSIONS: Some modifications of the rFVIIa molecule may not translate into a prolonged hemostatic effect.

Determining the impact of instrument variation and automated software algorithms on the TGT in hemophilia and normalized plasma.

BACKGROUND: Despite increasing recognition as a more precise test of in vivo hemostatic conditions, standardization of the thrombin generation test (TGT) continues to hinder its development as routine clinical practice. Prior efforts largely focused on comparing the effects of experimental conditions and different reagents. Commercialized kits, instruments and software have been introduced to calculate the TG curve and its parameters. However, modified versions of the TGT continue to be used worldwide on a variety of microplate reader instruments and processed using individualized algorithms. No prior study has compared the effect of instrument choice and its inherent noise profile on the processing of the TG curve and its common endpoint parameters. MATERIALS AND METHODS: Hemophilia A plasma supplemented with buffer or Factor VIII, mimicking hemophilic or normalized samples respectively, was monitored for thrombin generation after activation with TF on six different fluorescent microplate readers. Each instrument was optimized for TGT signal recording prior to testing. An automated software package containing various mathematical algorithms was utilized to compute the TG curves and parameters, and compare different TG processing approaches. RESULTS: Instruments produced unique noise profiles and end-point parameters that were incomparable in absolute signal terms. Similar relative hemophilic responses were obtained across various instruments when the normalized plasma sample was used as an internal standard. Smoothing algorithms corrected destructive instrument noise. CONCLUSIONS: Instrument-induced errors from numerical differentiation during TG curve processing cannot be eliminated by external calibrators, and require careful qualification of the instrument and implementation of noise-reducing software algorithms.

[Shunt surgery with reference to indications for liver transplantation]. / Shuntchirurgie im Hinblick auf die Indikation zur Lebertransplantation.

Sclerotherapy and protosystemic shunt surgery are established procedures for treating bleeding esophageal varices. Liver transplantation (OLT) has not yet been established as primary therapy of portal hypertension. In a series of 43 patients with uncontrollable bleeding, shunt surgery was employed in 26 patients with contra-indications to OLT and in 2 whose liver disease had not progressed. In the remaining 15 patients, OLT was performed in urgent (n = 8) and emergency settings (n = 7). The results obtained indicate that immediate shunting in nontransplant candidates results in one-year survival for 67-83% of Child's class A or B Patients and only 42% Child's C category regardless of the type of shunt procedure performed. In transplant candidates with a similar risk status (Child's B and C) 5 of 8 patients receiving an urgent transplant and 4 of 7 patients receiving emergency OLT during active bleeding survived for one year or more. These results indicate that bleeding varices can be treated successfully by OLT.