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  • br Development of calibrators and controls

    2022-06-29


    Development of calibrators and controls for DiXaIs
    Method validation and comparison with reference technique (LC:MS) At this stage, anti-Factor Xa bioassays can be established for reliably measuring DiXaI concentrations in plasma, with the available appropriate drug specific calibrators and controls. The next step requires validation and verification of the assays' performance characteristics, and demonstration that the adequacy and accuracy of drug measurements at clinical laboratory sites, over all the requested concentration range which can be observed in current practice, i.e. high levels if suspicion of overdosage or progressive drug accumulation (deficient clearance), or need to know concentrations at peak or at through (therapeutic compliance or verification of drug kinetics), or request for drug measurements in emergency (occurrence of bleeding or thrombosis in patients treated with DiXaIs), and especially before surgery [53], [54]. Recent reports show that patients anticoagulated with DOACs tend to have delayed surgery as compared to non-treated patients [22]. Measurement of drug concentration in cisapride is necessary for verifying that it is low enough for safely undertaking surgery, without the additional bleeding risk associated with a too high residual drug concentration [27]. Availability of drug specific antidotes will be of great usefulness in this context as it will allow neutralization of excess drug before surgery, and make it possible to undertake surgery without any additional delay due to the wait for drug clearance [20], [47], [51]. For verification and validation studies, first the assays' performances are established. In order to correctly and accurately measure concentrations in the high range (when needed), up to 500 ng/ml or above, or in the low range, at about 30 ng/ml, especially before an emergency surgery in treated patients, two assay ranges are designed: the standard range from about 25 to 500 ng/ml; a more sensitive range from about 5–10 ng/ml up to 100 ng/ml (Fig. 5). Both ranges use a similar assay design and instrument set-up, with different calibrators and a different tested specimen dilution. This approach with 2 assay ranges (Fig. 5) avoids any matrix effect which could result from the use of different plasma dilutions according to the expected DiXaI concentrations. The Anti-Factor Xa assays can be used with any of the automated laboratory instruments available, with instrument-specific settings. The Lower Limit of Quantitation (LLOQ) can present some variations from instrument to instrument, but it is of about 20–40 ng/ml for the standard range and of 5–10 ng/ml for the low range. Comparison with the LC:MS reference method is done through the use of clinical samples, obtained from drug clinical studies or from treated patients. Different articles have reported an excellent correlation between the LC:MS method and the 1-stage (kinetics) or the 2-stage Anti-Factor Xa bioassays [43], [44], [46], validating the in house results obtained (Fig. 6). There is an excellent correlation between the different types of methods, and measured values are fully comparable. Both Anti-Factor Xa bioassays are then appropriate for reliably measuring DiXaI concentrations in plasma, for all the concentration range expected, i.e. high or low concentrations. Independent reports confirm this good compliance between Anti-Factor Xa bioassays and the LC:MS method for measuring DiXaI concentrations in patient plasmas [44], [46].
    Regulatory requirements and method registrations
    Pre-analytical requirements for DiXaIs measurements DiXaIs can be measured in a standard citrated blood collection, using plasma prepared in compliance with CLSI guidelines or with country-specific recommendations. Usually 0.109 M citrate anticoagulant is the most frequent plasma preparation used for measurement of DOACs (Table 2). However, use of 0.129 M citrate or EDTA anticoagulated plasma preparations is possible as assayed plasma is always tested diluted, and anticoagulant has no interference with these anti-Factor Xa chromogenic assays. When comparing DiXaI measurements performed with bioassays to the LC:MS method, it is necessary to measure drug on the same anticoagulated sample in order to avoid any bias due to a different dilution factor induced by anticoagulant (citrate introduces a higher sample dilution than EDTA). Plasma is prepared from citrate anticoagulated blood through a 15 min centrifugation at 2,500 g, or with the usual centrifugation force used in the laboratory, hence no special caution is needed. Platelet poor plasma is used, but residual platelets have no interference in the assay, as could be the case for the assay of UFH or LMWH or LA. Plasma obtained is stable for several hours on the bench, and it can be stored frozen at <20 °C for at least 6 months (and probably much longer) without any loss of DiXaI concentration measured, either with LC:MS or with bio-assays.