71 Comparing FVIII concentration and activity FVIII plasma concentration with LC-MS/MS The FVIII plasma concentration was measured using the LC-MS/MS method at the ISO15189-certified Laboratory of Clinical Pharmacy. The LC-MS/MS method development is extensively described in the previously published manuscript of El Amrani et al. and was validated in accordance with European Medicines Agency guidelines on bioanalytical methods [16]. In brief, the method starts with the dissociation of the Von Willebrand factor from FVIII by triggering the coagulation cascade, which generates an unbound FVIII molecule. Subsequently, FVIII is selectively extracted by immunoaffinity interaction using monoclonal anti-FVIII camelid nanobodies. After washing, the FVIII is eluted, heat denatured, and trypsin digested. Finally, a specific peptide sequence from the A3 active domain of the FVIII molecule is selected as a surrogate for quantification by mass spectrometry. This signature peptide has a sequence that is unique for FVIII proteins, both endogenous and exogenous, and the sequence is not present in other human proteins. The method had a mean precision (%CV) within-run of 6.8% and between-run of 7.2%, a mean accuracy (bias%) of -2.6%, a linearity range 1–500 ng/mL, and 1 ng/mL was the lower limit of quantification. Determinants and patient characteristics Patient-, disease-, and treatment characteristics, such as gender, age, weight, exogenous FVIII products, anti-FVIII antibodies (also termed inhibitors), and co-medication interacting with the coagulation cascade, were identified as potential determinants for differences between the two methods. Anti-FVIII antibodies were measured with the Bethesda assay (Nijmegen modification) for which the clinical cut-off ≥0.6 Bethesda Units (BU) per millilitre was used [14]. Samples were carefully checked for presence of exogenous FVIII products by (1) all comments linked to the assay orders (2) data on active medication obtained from the Research Data Platform (also taking drug half-life into account) (3) multiple measurements of activity and inhibitors per patient in time. Based on this information the samples were labelled as ‘exposed’ or ‘unexposed’ to exogenous FVIII product, independent of the presence of endogenous FVIII or procoagulant comediation. The information regarding the determinants was obtained from medical records using the Utrecht Patient Oriented Database, a local data platform [15]. Data analysis To enable statistical analyses on laboratory results expressed in different units and with categories of different interval size, the correlation between FVIII activity and plasma concentration was evaluated with linear regression and a Bland-Altman analysis. The potential determinants of relative differences were analysed with linear regression analysis. The differences in the Bland-Altman analysis and in the linear regression of the determinants were expressed as relative differences not only to compare the two parameters of different units but also to compare the differences over the complete activity range (since an absolute difference in the lower range would weigh less compared to an absolute difference in the higher activity category). For the interpretation of the 4
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