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  • br Interference in coagulation assays

    2021-11-30


    Interference in coagulation assays Conversely to heparin, or heparin like Tenofovir Disoproxil Fumarate (LMWH, Fondaparinux, Sodium Danaparoid), which are catalytic inhibitors requiring the presence of AT for their activity, as depicted on Fig. 2, DiXaIs are directly targeted to Factor Xa, and are reversible [39]. DiXaI drugs have variable inhibitory constants for Factor Xa. This Factor Xa is generated at the crossroads of the intrinsic and extrinsic coagulation pathways, and therefore its inhibition impacts most of the coagulation tests in a variable manner according to the drug characteristics [32], [34]. In these laboratory methods, the Factor Xa generation kinetics are slower and the DiXaI interference is higher, which explains the different effect on the various coagulation assays. DOACs interfere in the global clotting assays, prothrombin time (PT) and activated partial thromboplastin time (APTT). This interference can be very different from reagent to reagent, and they present highly variable sensitivities to DOACs. Fig. 3 shows, for 4 different PT reagents (designed with human or rabbit Tissue Factor), and, for 3 different APTT reagents, the dose–response prolongation of clotting times. Increasing concentrations of DiXaIs (Rivaroxaban or Apixaban are tested) prolong the PT clotting time in a variable manner, and reagents with human recombinant Tissue Factor (TF) (Innovin®) have a higher sensitivity than those designed with rabbit extracted TF (Neoplastin®). Interestingly, the DTI drug, Dabigatran, was reported to have a stronger effect on the APTT than on the PT, and DiXaIs, especially Rivaroxaban, on APTT. Apixaban has little effect on both PT and APTT, and concentrations associated with a bleeding risk can occur in the presence of a PT or APTT within the normal range [20]. Our experience (Fig. 2) confirms this lack of sensitivity of global assays to Apixaban. Therefore, the global assays, PT or APTT, are not recommended for quantitatively measuring DOAC anticoagulant activity in plasma, although they can have some usefulness as screening methods in the presence of high drug concentrations [34], [40], [41], [42], [43], [44]. In addition to reagent variability, these global assays are sensitive to the patients' plasma factors, especially in disease states, and this induces lack of specificity for correlating clotting time prolongation with drug concentration. Some authors propose that global assays can be used only for “detecting” high DOAC concentrations in plasma when reagent sensitivity is duly documented: APTT for Dabigatran (DTI) and PT for Rivaroxaban [20]. No global assay can be used for Apixaban, due to the lack of sensitivity of these methods for this drug. Among the major indications for DiXaIs (or generally DOACs) measurements in plasma, is before an emergency surgery, and, especially, when it is associated with a high bleeding risk: the presence of residual DOAC concentrations above a defined threshold (30 ng/ml) must be excluded, and low concentrations need to be accurately and quantitatively measured in plasma [27], [28]. Highly sensitive, accurate, quantitative and easy to use assays are needed. For measurement of DiXaIs in plasma, anti-Xa methods are then the methods of choice, and they have to be run with drug specific calibrators and controls [38], [39]. This will be discussed in the upcoming paragraphs. DOACs in general, and DiXaIs in particular, can also interfere in most of the coagulation assays. The laboratory must be aware of this possible interference when treated patients are addressed for coagulation testing, or in the presence of an unexpected clotting time prolongation in unknown samples. This interference occurs with many clotting assays, but also with some chromogenic assays [34]. DiXaIs are expected to have a special impact on all assays where coagulation or activity goes through Factor Xa generation, or uses Factor Xa, whilst no interference is expected in thrombin based assays, such as the Thrombin Time (TT), Ecarin Clotting Time (ECT) or fibrinogen measurement with the von Clauss method. DiXaI interference can then be observed in many clotting or chromogenic assays for coagulation factors or inhibitors. However, the higher the plasma dilution used for the assay, the lower is the expected interference, although this general concept is not always observed in practice. As shown in Fig. 4, we noticed this interference in clotting tests for coagulation factors (using deficient plasmas and the PT or APTT method), and especially for measurement of Factors VIII and IX. There is also interference with the clotting assays designed for APC-R (activated Protein C Resistance/Factor V-Leiden), Protein S or Lupus Anticoagulant (LA), although in LA clotting assays this impact is variable from reagent to reagent. DiXaI interference is observed to a lesser extend for the Protein C clotting assay and for the chromogenic assays of Antithrombin (AT), although it is based on Factor Xa inhibition. The presence of DiXaIs significantly impacts chromogenic assays for Factor VIII, Factor IX, Factor X, for example. As an example of dose dependent incidence of DiXaIs in laboratory assays, Fig. 4 reports the interference of Rivaroxaban and Apixaban in the Protein S and Factor V-Leiden (APC-R) coagulation assays and in the Factors VIII and IX chromogenic assays. This interference is much weaker for the Protein C clotting assay. Fibrinolysis assays are not impacted as they do not use Factor Xa or Thrombin. No interference is observed in the chromogenic assays for Plasminogen, Alfa 2 – Antiplasmin, PAI-1, or tPA, or for the various Latex based immuno-assays, either using agglutination or automated photometric detection, or for ELISAs.