78 Chapter 4 0.8–5.3 BU/mL, n = 5) present, we found a substantial higher FVIII plasma concentration than activity. Due to the small sample size of anti-FVIII antibodies in this study, the proportional correlation between antibody level and relative difference could not be confirmed. This result might indicate that for some patients the specific FVIII-antibody complexes are still present in the circulation, not being cleared by the immune system, and that non-functional FVIII is still being measured by the LC-MS/MS method. This phenomenon deserves further clinical exploration, but could not be included in the present study as bleeding data were unavailable for this retrospective study on remnant material. Another essential finding of this study is the trend seen in exogenous FVIII products of a higher FVIII activity than plasma concentration. This result is consistent with the study of Barrowcliffe et al. mentioning an overestimation of FVIII activity by unbound FVIII, since FVIII may become activated during sample collection [23]. Other reasons for overestimation of FVIII activity are that OSA is simply not validated for innovative new products such as shortened (turoctocog alfa) or deleted (efmoroctocog alfa) B-domain modified products or Fc-fusion (also efmoroctocog alfa) products, and also the discrepancy seen in mild and moderate patients with haemophilia A [5, 12, 24]. The LC-MS/MS method is less affected by the new types of FVIII products since the camelid antibodies have a high affinity for the specific FVIII epitope. It should be mentioned that the full-length FVIII product group was on borderline of significance, which might make this group interesting as well to investigate in a study with more power. Limitations and strengths The study was limited by the retrospective design and the lack of an appropriate gold standard to compare to the new LC-MS/MS method. Although OSA is a functional assay with result variability, OSA was used as a reference in this study, since it is the most used method worldwide, and corrected for by using relative differences in this study [25]. Bias might have been introduced by including the same patient in another category, but a sensitivity analysis demonstrated no differences in results. We performed this study as a proof of principle experiment to commence validated further clinical research on FVIII quantified with mass spectrometry, therefore undoubtedly making the strength of this study its first clinical contribution. Recommendations Further research regarding the quantification of FVIII with OSA, CSA, and LC-MS/MS is indicated and is expected to start at our center to make a well-founded recommendation for the potential correcting steps per different subgroups of patients and products and for correcting steps to simplify the interpretation of the FVIII plasma concentration. A first recommendation for future research would be to conduct a prospective study where the FVIII activity (OSA and CSA) and FVIII plasma concentration (LC-MS/MS) is measured and compared in multiple samples over time, in a curve, after administrating
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