As for all DOACs measurement of

As for all DOACs, measurement of DiXaIs activity requires the use of dedicated specific calibrators and controls. Each drug needs its own calibration and control material in a like to like manner. In no case can a drug concentration be extrapolated with the use of a calibrator established for another drug. More especially, the usual Anti-Factor Xa International Units (IU), established for LMWH and UFH and available with WHO International Standards (which can be obtained from NIBSC, South Mimms, UK), cannot be used for standardization of Factor Xa inhibitory units. Actually, heparin (indirect catalytic inhibitors) Anti-Factor Xa International Units are quite differently defined than those for the direct inhibitory activity of DiXaIs. Practically, DiXaIs are calibrated with dedicated AT13148 calibrators prepared by spiking an accurate known amount of solubilized drug in plasma, and preparing a calibration range through dilutions in a normal citrated plasma pool, covering all the assay dynamic range. In fact, for commercial reagents DiXaI drugs are spiked at various concentrations in a citrated normal plasma pool, distributed at an exact concentration in siliconized vials, and lyophilized. Usually, 1.00 ml of drug supplemented plasma pool is lyophilized into vials in the presence of stabilizers, and these vials have to be restored with 1.00 ml of distilled water before use. The drug concentration in the restored vial is always slightly below the one spiked in plasma, mainly because when lyophilized plasma is restored with 1.00 ml distilled water (i.e. the same volume used for distributing supplemented plasma into vials and freeze drying) the volume of restored plasma is slightly higher than 1.00 ml. This is the effect of the lyophilized plasma pellet volume, including the lyophilization excipients used, which produces a final volume which is about 8–10% increased compared with the volume before lyophilization. Calibrator sets established for the various DiXaI drugs can then be available for calibrating the Anti-Factor Xa assays used. Drug concentrations in restored lyophilized vials are expressed in ng/ml. The key issue is to have a reliable reference material for the exact value assignment of drug concentration in lyophilized vials. No International Standard material is available for these drugs, and therefore a robust reference has to be established. This is done through a close collaboration with the pharmaceutical industry, in order to have access to the active DiXaI material. This cooperation with pharmaceutical companies allows access to well characterized drug preparations, and accurately documents the lyophilized calibrator sets prepared. Value assignment for drug concentration is accurately defined for an internal reference lot (Internal Standard) using the LC:MS method. Although LC:MS method measures mass concentration, and Anti-Factor Xa assays the anti-Factor Xa inhibitory activity, there is an excellent compliance between both types of assays, as almost all the drug present in plasma keeps its full activity. Actually, for establishing a robust enough reference material, a multi-step approach is performed. The first prototype lot is prepared and a value assignment of DiXaI concentration is done by LC:MS, and, only for documentation, it is also measured with the Anti-Factor Xa methods using DiXaI freshly spiked plasmas for calibration (which allows to confirm the variation between drug concentration in plasma before and after lyophilization). Only the LC:MS value is kept for this 1st prototype lot. Then, a second prototype lot is prepared and DiXaI concentrations are established in blind using the LC:MS method or the Anti-Factor Xa bioassays calibrated with the 1st prototype lot and LC:MS assigned values. Concentrations obtained with both methods are then matched, and good compliance is obtained between both approaches, with a bias which is <5%. For example, Table 1 shows for this 2nd prototype lot and for Apixaban, the comparison between the measurement in blind of drug concentrations using the LC:MS method or both Anti-Factor Xa bioassays: the kinetics (1-stage) or the 2-stage methods. In order to limit the own variability impact of the LC:MS method itself, a 3rd prototype lot is prepared and DiXaI concentrations are again measured in blind with LC:MS or the Anti-Factor Xa bioassays, using in this latest case two independent series: the first one being calibrated with the 1st prototype lot (with LC:MS assigned values) and the 2nd one with the 2nd prototype lot (and also the LC:MS assigned values). Mean concentrations obtained with both series of measurements are averaged and compared with LC:MS concentrations. This 3rd prototype lot becomes then the Internal Standard which will be used for DiXaI concentration assignment for all the upcoming manufactured lots. This approach allows preparing a robust reference material and ensuring a good reproducibility and consistence of assigned values over time. When a new internal preparation is needed, verification of values assigned with bioassays is done with the LC:MS method in order to prevent any variation on time.