Dogne, Jean-Michel

Douxfils, Jonathan

Evrard, Jonathan

Mullier, François

Siriez, Romain

English

Repository of the University of Namur

RESEARCH OUTPUTS / RÉSULTATS DE RECHERCHEAuthor(s) - Auteur(s) :Publication date - Date de publication :Permanent link - Permalien :Rights / License - Licence de droit d’auteur :Bibliothèque Universitaire Moretus PlantinInstitutional Repository - Research PortalDépôt Institutionnel - Portail de la Rechercheresearchportal.unamur.beComparison of the impact of DOACs on hemostasis diagnostic testsSiriez, Romain; Evrard, Jonathan; Dogne, Jean-Michel; Mullier, François; Douxfils, JonathanPublished in:Comparison of the impact of DOACs on hemostasis diagnostic testsPublication date:2019Link to publicationCitation for pulished version (HARVARD):Siriez, R, Evrard, J, Dogne, J-M, Mullier, F & Douxfils, J 2019, Comparison of the impact of DOACs onhemostasis diagnostic tests. in Comparison of the impact of DOACs on hemostasis diagnostic tests.General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            • Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            • You may not further distribute the material or use it for any profit-making activity or commercial gain            • You may freely distribute the URL identifying the publication in the public portal ?Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Download date: 23. Jun. 2020Comparison of the impact of DOACs on hemostasis diagnostic testsRomain Siriez : romain.siriez@unamur.be Tel. : (+32)81.72.43.25 Rue de Bruxelles, 61, 5000 - NamurRomain Siriez1, Jonathan Evrard1, Jean-Michel Dogné1, François Mullier2, Jonathan Douxfils1,31 University of Namur, Department of Pharmacy, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for LIfe Sciences (NARILIS), Namur, Belgium; 2 Université catholique de Louvain, CHU UCL Namur, Hematology Laboratory, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for Life Sciences (NARILIS), Yvoir, Belgium; 3 Qualiblood s.a., Namur, BelgiumInterpretation of hemostasis diagnostic tests results should be done with caution in patients on DOACs since many of them may be impacted. The rational use of appropriatereagents for diagnostic tests must be performed according to the present DOAC for antithrombin level measurement or activated protein C resistance. For clotting factors levelsmeasurements, the use of DOAC-insensitive PT or aPTT reagents with adapted dilution of samples should be considered. Finally, the use of a device/chemical compound able toremove or antagonize the effect of DOACs or the development of new diagnostic tests insensitive to DOACs should be considered to further minimize the risk of false results forlupus anticoagulant.ConclusionBackground and aimThere is a laboratory and clinical need to know the impact of anticoagulants on diagnostic teststo avoid misinterpretation of results. Although the labeling documents of these medicines (i.e.EMA summary of products characteristics, FDA prescribing information) provide someinformation about the influences of each DOAC (factor Xa inhibitors including apixaban,edoxaban, rivaroxaban and betrixaban and factor IIa inhibitor dabigatran) on diagnostic tests,these are usually limited to some of the most common tests, with few information about thereagents used and not head to head comparison. This study aims to compare the impact ofDOACs on a large panel of hemostasis diagnostic tests and provide practical recommendationsfor clinicians.MethodsThe impact of increasing concentrations of DOACs on a normal pooled plasma has beenassessed on several thrombophilia testing, including assays for antiphospholipidsyndrome, protein C, protein S and antithrombin activity, measurements of clottingfactors levels and the detection of activated protein-C resistance with a large panel ofreagents. The results are compared and discussed with data obtained from theliterature.Overall, factor Xa and factor IIa inhibitors significantly affect clot-based hemostasis diagnostic tests resulting in false-positive or false-negative results. Impacts on thrombin-based hemostasisdiagnostic tests are observed with dabigatran but not with anti-Xa and conversely for FXa-based hemostasis diagnostic tests (Figure 1-2). No impact was observed with antigenic or chromogenicmethods for the assessment of protein S and C activity (Data not showed).Significant impacts of DOACs on hemostasis diagnostic testsFigure 1: Impact of factor Xa inhibitors related to indications on diagnostic assays. Laboratory testing of factor Xa inhibitors and theirimpacts on diagnostic assays. Red and blue lines represent plasma concentrations at peak and trough in NVAF and VTE, respectively. Green boxesrepresent ranges of concentrations that can impact diagnostic assays and lead to misdiagnosis while grey boxes represent unaffected assays. Forclotting factors measurement, boxes represent the dynamic range of quantitation (the dynamic range of quantitation is defined as range covering fromthe lowest observed limit of quantification to the maximal concentration tested or supposed) of PT and aPTT for sensitive reagents. Ctrough: Minimumplasma concentration during the dosing interval; Cmax: maximum plasma concentration during the dosing interval; VTE: Venous thromboembolism;NVAF: Non-valvular atrial fibrillation; OD: once daily; bid: twice daily; APCR: Activated Protein C Resistance. Notes: Data on plasma concentrationswere extracted from J. Douxfils et al. for apixaban, edoxaban, rivaroxaban and from R. Siriez et al. and SmPC for betrixaban.Figure 2: Impact of factor IIa inhibitors related to indications on diagnosticassays. Laboratory testing of factor IIa inhibitors and its impacts on diagnostic assays. Redand blue lines represent plasma concentrations at peak and trough in NVAF and VTE,respectively. Green boxes represent ranges of concentrations that can impact diagnosticassays and lead to misdiagnosis while grey boxes represent unaffected assays. For clottingfactors measurement, boxes represent the dynamic range of quantitation (the dynamicrange of quantitation is defined as range covering from the lowest observed limit ofquantification to the maximal concentration tested or supposed) of PT and aPTT forsensitive reagents. Ctrough: Minimum plasma concentration during the dosing interval;Cmax: maximum plasma concentration during the dosing interval; VTE: Venousthromboembolism; NVAF: Non-valvular atrial fibrillation; OD: once daily; bid: twice daily.Notes: Data on plasma concentrations were extracted from Douxfils et al., Favaloro et al.,Gessoni et al. and Bonar et al.Providing recommendations to deal with impacts of DOACs on hemostasis diagnostic tests is a clinical challenge since these impacts depend on i.) the anticoagulant agent; ii.) the plasma level of anticoagulant and iii.) the sensitivity of the tests and the reagents/methodologies used. - Lupus Anticoagulant: LA are immunoglobulins binding phospholipids and protein providing a disruption of coagulation process prolonging phospholipid-dependent coagulation tests, as APTT. Recommendation is to use two different tests for the detection of LA such as a sensitive APTT assays or a dilute Russell viper venom tests (dRVVT) :- aPTT : aPTT with low phospholipids (PL) concentrations making them very sensitive to LA is performed with a classical aPTT-based test with higher amount of PL to correct the clotting time by saturating the antibodies. - DRVVT : The venom from Daboia russelii is used as an activator of the endogenous factor X presents in the sample. The test is performed with two different concentrations of added phospholipids. A screen test is first performed with a low concentration of phospholipids to suspect the presence of LA antibodies.  If the screen ratio between the  patient’s clotting time and a reference pool plasma is at least equal to 1.2 then a confirmatory test, with high concentration of phospholipids, is performed against versus the reference pool plasma. An impact on both tests could be observed since these tests required FXa and FIIa. Furthermore, an impact on diagnostic is explained by an increased sensitivity of thescreen reagent due to the low concentration of PL. The presence of DOAC will impact results of screen and confirm assays in different ways that will provide a ratioscreen:confirm higher than 1.2 inducing unnecessary investigations.- Activated Protein C Resistance: Several assays exist to assess the APC-R:- The first assay used the ratio between baseline aPTT and aPTT in presence of purified exogenous APC to inactivates factor Va and factor VIIIa.  The presence of APC results in an increased clotting time of the APTT in samples without FV Leiden mutation. By contrast, in a sample with mutation the prolongation in theclotting time is less marked. Nevertheless, any factors that could impact aPTT assays (LA, anticoagulants treatments, …) could false this interpretation. - Second assays improved sensitivity and specificity by diluting plasma sample with FV-deficient human plasma (1:4) but was still influence by LA and oral anticoagulants. - Third generation assay used Russell’s viper venom from Daboia Russelli to overcome the influence of contact pathway but without dilution in FV-deficient human plasma, low FII, FV as well as fibrinogen concentrations, anticoagulants may interfere. - Finally, researchers developed plasma based functional clotting assay with the replacement of FXa by noscarin, a FVa-dependent and phospholipid-independent prothrombin activator isolated by Notechis scutatus scutatus. A dilution with FV-deficient plasma (1:4) is performed to overcome to factor deficiencies/elevations and the absence of phospholipids in the reagent avoid impacts of lupus anticoagulants. On one hand, the assessment of APC-R based on aPTT and using Factor V-deficient plasma seem to be impacted by DOACs contrary to prothrombinase-based assay as the Pefakit APC-R factor V Leiden which is only impacted by edoxaban and dabigatran, due to their impact on thrombin, according Douxfils et al. and Favresse et al. On the other hand, Hillarp et al. showed that edoxaban do not impact Pefakit ACP-R factor V Leiden. Theses contrary results could be explained by the high lot-to-lot variability of phospholipids concentrations or by theslight anti-thrombin activity of edoxaban.- Antithrombin level measurement: An assessment of antithrombin activity can be performed by immunological or functional assays. Functional AT assays are chromogenic assays based on the principle of FIIa (human or bovine) or FXa (human) inhibition. The antithrombin measurements using FIIa-based chromogenic assays (e.g. Berichrom ATIII® or Stachrom ATIII®) are not influenced by direct Xa inhibitors (apixaban, edoxaban, rivaroxaban and betrixaban) and the antithrombin measurements using FXa-based chromogenic assays (e.g. Coamatic LR® or HemosIL® Liquid Antithrombin) are not influenced by dabigatran. Immunological assays are not impacted by the presence of DOACs.- Clotting factors levels: The evaluation of factors from extrinsic pathway (FVII, FX, FII, FV) use a PT-based clotting method unlike the evaluation of intrinsic pathway factors (FVIII, FIX, FXI and FXII) which required an aPTT-based clotting method. Given the fact that chronometric tests are severely impacted by DOACs, the presence of one of these drugs impact the measurement of clotting factors leading to an underestimation of the levels of clotting factors. Although the impact is less pronounced when the dilution is high, a significant decrease in the accuracy of the test is noticed. Moreover, the measurement of clotting factor should be performed with insensitive PT- or aPTT-reagents , or known to be less sensitive (e.g. Dade®Innovin® for PT and C.K-Prest® for aPTT are less sensitive to the presence of DOAC). The ionic force, the pH of the buffer solution, the source of activator or even the phospholipids composition could be parameters that impact on the sensitivity of a particular assay.

Comparison of the impact of DOACs on hemostasis diagnostic tests

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Comparison of the impact of DOACs on hemostasis diagnostic tests

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