77 Comparing FVIII concentration and activity DISCUSSION This study demonstrates that regardless of an overall strong correlation, there is a large variability between FVIII activity measured by OSA and the FVIII plasma concentration measured with LC-MS/MS. Significant differences between the two methods were independently correlated with the presence of anti-FVIII antibodies or use of exogenous FVIII products. Correlation and variability To our knowledge, this is the first clinically established comparison between a FVIII activity assay and a FVIII plasma concentration method using LC-MS/MS. A strong correlation was expected, however, we did not expect this large variability. Explanations for the large variability could be (i) the influences of the determinants and (ii) the OSA result variability. We found that the FVIII plasma concentration measurements are evidently higher compared to the FVIII activity in the high measurement range (from >40). As only haemophilia A patients were included, activity results >40 IU/dL (cut-off for diagnosis) could be indicative for the presence of FVIII product. When hypothesizing, a potential false estimate of FVIII activity in this range could result in a misleading drug half-life, which might be interesting to investigate with efficacy outcome measurements in new upcoming research [8]. Another explanation for a higher plasma concentration than activity is that LC-MS/MS method may measure dysfunctional FVIII, with no activity, in so called cross-reacting material (CRM) positive patients [16]. In contrast, in the range of <1– 40IU/dL FVIII activity, most measurement points were under the regression line or relative difference line (indicating a higher activity than plasma concentration). In other studies, OSA demonstrated a significantly higher activity than CSA in approximately 30% of the moderate and mild patients with haemophilia A [17, 18]. To prevent misclassification in the diagnostic phase, but especially to prevent underestimation of the bleeding risk, it is preferred to use more than one OSA activity measurement, to combine OSA with CSA, or to identify the FVIII gene mutation [19, 20]. Whether a FVIII plasma concentration measurement is a better representation of the clinical effect than the FVIII biological coagulation activity cannot be demonstrated with the results of this exploratory study. The presented data support the supposed overestimation of OSA in selected samples, which might indicate that LC-MS/MS could be a useful predictor for classification of disease severity and therapeutic monitoring in these specific patients, but this needs to be confirmed with further research. Determinants Currently, the major complication in haemophilia A treatment is the development of neutralising anti-FVIII antibodies, rendering endogenous and exogenous FVIII ineffective. Anti-FVIII antibodies neutralise the FVIII activity by forming FVIII-antibody complexes that accelerate clearance of FVIII or by sterically hindering the interaction of FVIII with other coagulation factors [21, 22]. For the samples with anti-FVIII antibodies (in the range 4
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