Anouk Donners

51 LC-MS/MS-method development quantifying FVIII INTRODUCTION Congenital haemophilia A is a clotting disease caused by a defect in the factor VIII (FVIII) gene located on the X-chromosome and therefore predominantly affects the male population. The prevalence of haemophilia A is 1 in 5.000 male persons. Haemophilia A can lead to serious complications such as bleeding after surgery, bruising, disabling arthropathy, gastrointestinal and urological bleeding and intracranial hemorrhage [1-5]. Acquired haemophilia A is rare and has been estimated to affect 0.2 – 1 in 1 million persons per year [4]. Acquired haemophilia A is caused by the development of autoantibodies directed against FVIII [4, 6-8]. The majority of the patient population (60%), which consists predominantly of men around the age of 60 years, have a severe type of haemophilia A with FVIII activity usually below 3%. The current treatment requires infusion with recombinant or plasma-derived FVIII [9]. Furthermore, patients with congenital haemophilia are also at risk of developing inhibitors against FVIII. Some of these patients also require high dose FVIIa, which may increase the risk of thrombosis. Patients with either congenital or acquired haemophilia A are diagnosed and treated depending on the activity of FVIII in plasma and therefore careful monitoring of these patients is critical [10]. Currently, there are three commercially available assay types for measuring FVIII activity: a one-stage assay, a two-stage assay, and a chromogenic (Amidolytic) assay [11-14]. The most commonly used assay is the one-stage assay, which is based on activated partial thromboplastin time (aPTT) [15, 16]. This assay measures the time required for patient plasma to form a fibrin clot after it has been combined with a FVIII deficient plasma and reagents to initiate clotting have been added [17, 18]. The one-stage assay is relatively fast and cheap to perform on automated coagulation analyzers. However, this assay has been shown to have higher %CV for elevated FVIII values in comparison to the other methods [14, 19]. Depending on the endpoint detection method, the one stage-assay may be more sensitive for lipemic plasma samples, and the assay may also have varying sensitivity and specificity to anticoagulant medication and to endogenous inhibitors such as antibodies against FVIII or lupus anticoagulant. Depending on the dilution factor and the activator used in the one-stage assay, both of these types of antibodies may lead to a prolonged aPTT value, which would result in a low FVIII activity, hence complicating diagnosis [20, 21]. Furthermore, due to the lack of standardization in instrumentations and reagents the intra-laboratory variation is the highest in this type of assay [11]. Finally, some patients with mild or moderate haemophilia A will show correct lower activity in the chromogenic assay and two-stage assay but will show false normal activity with the one stage assay possibly leading to misdiagnosis [11, 22]. Aside from these technical limitations in the one-stage assay, activity-based assays in general are sensitive to sample condition. Citrate plasma kept at room temperature (25 °C) or at 4 °C needs to be analyzed within 2 to 6 hours after sample collection, due to the rapid degradation of FVIII, alternatively, the samples can be quickly processed and frozen at -80 °C [23-27]. In contrast to mass-spectometry methods, ligand binding assays pose other challenges such as cross reactivity and limited linearity. 3