Factor VIII (FVIII) activity of prophylactic replacement FVIII therapy used in patients with hemophilia A can be measured accurately by using chromogenic assays containing bovine factor X (FX) and either bovine or human factor IXa (FIXa) when the patient is on emicizumab, according to a study published in the International Journal of Laboratory Hematology. 

In addition to replacement FVIII therapy, emicizumab, a bispecific antibody, has emerged as a prophylactic treatment option for patients with hemophilia A, with or without inhibitors. Emicizumab differs from endogenous FVIII in that it binds to both active and inactive forms of factor IX (FIX) and FX; in addition, it can be administered without the preactivation/deactivation of the antibody. 

The assay most commonly used globally for measuring FVIII activity is the one-stage factor assay based on activated partial thromboplastin time (APTT). However, the use of this assay is discouraged if the patient is on emicizumab.

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“The interference that emicizumab has on some hemostasis assays is now well documented,” the authors of the study wrote. 

Another option is chromogenic FVIII assays that contain bovine FX and either bovine or human FIX proteins. These assays are insensitive to emicizumab and can be used to measure FVIII concentrations more accurately. 

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The authors of this study sought to measure FVIII activity with severe hemophilia A plasma that had been artificially spiked with emicizumab and replacement FVIII at various concentration levels. They compared the efficacy of the aforementioned assays. They also studied thrombin generation via 2 mechanisms: a traditional semi-automated tissue factor triggered assay and a fully automated system in the spiked plasmas. 

The results demonstrate that chromogenic assays containing bovine FX and either bovine or human FIXa can accurately measure FVIII activity, even in the presence of emicizumab. In contrast, one-stage assays overestimate replacement FVIII when emicizumab is present. Thrombin generation studies indicate that the combination of emicizumab and replacement FVIII therapy increases the generation of thrombin compared to replacement FVIII alone. 


Bowyer AE, Maclean RM, Kitchen S. The combination of emicizumab and recombinant factor VIII in plasma: which assays can we use for accurate measurementInt J Lab Hematol. Published January 29, 2023. doi:10.1111/ijlh.14021