Anouk Donners

162 Chapter 9 Potential future applications 1. Multiplexing in haemophilia A and beyond Regarding haemophilia A measuring and monitoring, the LC-MS/MS method could be used to multiplex. For instance, our research group developed a multiplex analysis of seven therapeutic monoclonal antibodies, including emicizumab, in human plasma or serum with a generic sample preparation [20]. Multiplexing can simultaneously measure multiple proteins in one run, which is helpful for diagnosing not only haemophilia A, but also other bleeding disorders (e.g., haemophilia B, VWF disease). This analysis could generate an entire coagulation profile or a ‘coagulation passport’ for an individual, which is especially advantageous for those with difficult venous access (e.g., children) because LC-MS/MS requires only 10 µL of plasma. A coagulation profile could also help individuals without a bleed history who present with unexpected and uncontrolled bleeding in the emergency room, although faster result reporting of LC-MS/MS is first required. Multiplexing could also be used to design an LC-MS/MS method to measure the entire therapeutic panel of drugs prescribed daily by a clinician. Thus, a specific ‘therapeutic haemophilia run’ could be developed that would be efficient from a logistical perspective in a clinical setting. The coagulation profile and the haemophilia A drug panel testing could even be combined in one run. However, the concentration-biomarker relationship of all the peptides in this run should be established because a quantifiable protein (i.e., concentration) does not necessarily mean that protein is functional (i.e., effect). This thesis began work on this topic in Chapters 3 and 4. 2. Cases of discrepant FVIII activity The monitoring moments and the treatment strategies are based on the traditional classification of severe, moderate and mild haemophilia A [1, 21]. This classification system was first described by Biggs and MacFarlane in 1958 and is still used in the ISTH SSC definition [22, 23]. The underlying FVIII gene mutation is often responsible for subnormal endogenous FVIII activity. Null mutations typically prevent FVIII-molecule synthesis and are associated with an undetectable FVIII activity, whereas non-null mutations permit the synthesising of some of the molecule and are usually associated with a residual FVIII activity [24]. The clinical distinction between severe and non-severe haemophilia A was confirmed in 2011 in a similar manner [9]. Although the classification generally relates to the frequency of bleeding symptoms, 10–15% of PwHA with the severe classification bleed less than expected [25-27]. This heterogenic phenotype in the severe classification is only partially explained by genetic and acquired factors [24]. About one-third of people with the non-severe classification demonstrate a discrepancy in the FVIII activity measurements between the one-stage clotting assay (OSA) and chromogenic substrate assay (CSA) [28, 29]. Most of these people display higher FVIII activity based on OSA when compared with CSA (standard discrepancy), whereas the opposite is true in a smaller group (inverse discrepancy) [30]. Depending on the specific