491Blood Transfus 2017; 15: 491-4 DOI 10.2450/2017.0301-16© SIMTI Servizi Srl

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Interference of direct oral anticoagulants in haemostasis assays: high potential for diagnostic false positives and false negatives

Emmanuel J. Favaloro1, Giuseppe Lippi2

1Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Sydney Centres for Thrombosis and Haemostasis, NSW Health Pathology, Westmead Hospital, Westmead, NSW, Australia; 2Section of Clinical Biochemistry, University of Verona, Verona, Italy

Many patients have a variety of prothrombotic risk factors or conditions, and these patients are often given anticoagulant/antithrombotic treatment. Such treatments for thromboembolic disorders are given to patients with atrial fibrillation or acute coronary syndrome, for prevention of venous thromboembolism in patients undergoing major, orthopaedic or cancer surgery or therapy and for prevention of recurrence in patients with previous episodes of venous thromboembolism1. Anticoagulant therapy has for decades been based on the administration of two main classes of drugs: vitamin K antagonists (e.g., warfarin) and heparinoids (unfractionated heparin, low molecular weight heparin, and other heparin-like molecules such as fondaparinux). These "classical anticoagulants" have proven effective for both primary and secondary prevention of systemic thromboembolism, but nonetheless have numerous drawbacks and limitations1-3, thereby paving the way for the introduction of new classes of anticoagulant/antithrombotic agents. These were originally termed new (or novel) oral anticoagulants (NOAC), and later (no longer being either new or novel) direct oral anticoagulants (DOAC); however, other terms have also been proposed, such as non-vitamin K antagonist oral anticoagulants (NOAC), direct specific oral anticoagulants (DSOAC) and target-specific oral anticoagulants (TSOAC)4,5.

DOAC have been designed specifically to inhibit directly either activated factor II (FIIa; dabigatran), or activated factor X (FXa; rivaroxaban, apixaban, edoxaban and betrixaban)4-7. DOAC have several important advantages over classical anticoagulants, making them favoured by clinicians and patients alike6-8.

An unfortunate downside to their popularity is under-recognition of their effects on laboratory tests by general clinicians. Indeed, the pervading "mantra" that laboratory monitoring is not required for patients taking DOAC may cause some clinicians to assume that the drugs do not affect haemostasis tests. They may thereby request a range of laboratory investigations on their "prothrombotic" patients, without even considering that these tests may be adversely affected by the drugs,

which many of their patients may be taking at the time of blood collection.

DOAC, like vitamin K antagonists and heparins before them, are particularly problematic in laboratory test practice, because of the timeline of causal events, given that: (i) patients are clinically assessed (e.g., in an emergency department) as having suffered some sort of "prothrombotic" event requiring anticoagulation, and are then (ii) quickly placed on some sort of anticoagulant therapy, and (iii) only afterwards "when the situation is considered more stable", is an investigation into why the patient suffered the initial event initiated4,5,9. However, once patients are on anticoagulant therapy, any ensuing attempt at laboratory testing for investigation of likely thrombophilia markers is earmarked for likely disastrous consequences. This is because in reality, contrary to the perception that DOAC do not affect haemostasis testing, most tests of haemostasis, including those commonly used to assess the prothrombotic status of a patient who has had a thrombotic event, are significantly affected by DOAC (Table I)5.

In another potential scenario, patients placed on DOAC therapy may have a bleeding event while on the drug, and may subsequently be investigated for laboratory markers of bleeding. This may occur either because of a disconnection between the patient and the clinician investigating the bleeding event (e.g., a patient presenting with trauma to an emergency physician), or because some preliminary investigation has uncovered an "unexpected" prolonged routine coagulation test result (e.g. activated partial thromboplastin time [APTT], or prothrombin time [PT]), and then more detailed investigations are undertaken (e.g., clotting factor assays) to help to explain the "initially unexpected" test result.

Table I provides a summary of the effects of DOAC on a wide range of haemostasis tests9. In brief, all DOAC affect routine coagulation tests such as PT and/or APTT, with dabigatran affecting the APTT more than the PT, and rivaroxaban affecting the PT more than the APTT. The thrombin time is very sensitive to dabigatran, but insensitive to the anti-FXa agents, and fibrinogen is usually not affected by any DOAC except for occasional

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Blood Transfus 2017; 15: 491-4 DOI 10.2450/2017.0301-16

Table I - Summary of DOAC effects on haemostasis assays (interference vs measuring).

Test Dabigatran Rivaroxaban Apixaban Comments

A. Routine assays (screening for DOAC or potential interference)

PT -/ ↑ ↑ / ↑↑ -/ ↑ Different reagents show different sensitivities; in general, order of sensitivity = rivaroxaban, dabigatran, apixaban; only a few reagents are sensitive to apixaban.

APTT ↑ / ↑↑ -/ ↑ -/ ↑ Different reagents show different sensitivities; in general, order of sensitivity = dabigatran, rivaroxaban, apixaban.

TT ↑↑↑ - - Very sensitive to dabigatran; insensitive to the anti-FXa agents.

Fibrinogen -/↓ -/↓ -/↓ Most von Clauss methods insensitive to all DOAC; occasional von Clauss methods and PT-based methods will show some false loss of fibrinogen.

dRVVT ↑↑ ↑↑ ↑↑ Most dRVVT methods are sensitive to all DOAC.

B. Quantifying assays (measuring levels of DOAC)

dTT/DTI ↑↑ - - Very sensitive to dabigatran; not affected by anti-FXa agents including rivaroxaban and apixaban.

ECT/ECA ↑↑ - - Very sensitive to dabigatran; not affected by anti-FXa agents including rivaroxaban and apixaban.

PICT ↑↑ ↑↑ ↑↑ Requires assay modifications to provide sensitivity to all agents; may require different set-ups for anti-FIIa vs anti-FXa drugs.

Anti-Xa - ↑↑ ↑↑ Sensitive to anti-FXa agents (including rivaroxaban and apixaban); insensitive to dabigatran.

C. DOAC interference (presence of DOAC will potentially yield false low or false high values)

PT coagulation factors

↓/↓↓ ↓/↓↓ ↓/↓↓ All PT coagulation factors affected by DOAC, although these are most sensitive to dabigatran and rivaroxaban; can also yield impression of (false) factor inhibitor.

APTT coagulation factors

↓↓/↓↓↓ ↓/↓↓ ↓/↓↓ All APTT coagulation factors affected by DOAC, although these are most sensitive to dabigatran and then rivaroxaban; can also yield impression of (false) factor inhibitor.

Protein C -/ ↑ -/ ↑ -/ ↑ Chromogenic tests unaffected; clot-based tests may be variously affected.

Protein S -/ ↑ -/ ↑ -/ ↑ Antigen-based tests (e.g., LIA) unaffected; clot-based tests may be variously affected.

Antithrombin -/ ↑ -/ ↑ -/ ↑ Anti-FIIa-based methods affected by dabigatran; anti-FXa-based methods affected by other DOAC.

APCr -/ ↑ -/ ↑ -/ ↑ May be variously affected; classically, APTT based assays mostly believed to be affected.

LA ↑ / ↑↑ ↑ / ↑↑ -/ ↑ LA tests are sometimes affected so that prolongation in screening assays exceed prolongation in confirmatory assays, leading to high screening/confirmatory assay ratios, and thus false determination of LA. This is especially true of dabigatran and rivaroxaban.

VWF - - - Not known to be affected by DOAC.

TEG ↓/↓↓ ↓/↓↓ ↓/↓↓ Various parameters affected.

LTA - * - - *Dabigatran may affect thrombin-induced aggregation; otherwise, not known to be affected by DOAC.

PFA - - - Not known to be affected by DOAC.

TGA ↓/↓↓ ↓/↓↓ ↓/↓↓ Various parameters affected.

mPT/dPT ↑ / ↑↑ ↑ / ↑↑ ↑ / ↑↑ Various modifications of the PT may increase sensitivity to all DOAC.

ACT ↑ / ↑↑ -/ ↑ -/ ↑ Similar to APTT.

ACT: activated clotting time; APTT: activated partial thromboplastin assay; APCr: activated protein C resistance; DOAC: direct oral anticoagulant; dRVVT: dilute Russell viper venom time; dPT: dilute prothrombin time; dTT: dilute thrombin time; DTI: direct thrombin inhibitor (assay); ECT: ecarin clotting time; ECA: ecarin chromogenic assay; LA: lupus anticoagulant; LIA: latex immunoassay; LTA: light transmission aggregometry; mPT: modified prothrombin time; PFA: platelet function analyser; PICT: prothrombinase-induced clotting time; PT: prothrombin time; TGA: thrombin generation assay; TT: thrombin time; TEG: thromboelastography; VWF: von Willebrand factor.

methodologies. In line with the relative sensitivities for PT and APTT, coagulation factors attributable to the intrinsic, extrinsic or common pathway are significantly affected by DOAC, with the APTT-based factors (VIII, IX, XI and XII) most affected by dabigatran10.

What is most relevant to the current editorial is the effect of DOAC on the thrombophilia tests sometimes used to investigate patients who have had a thrombotic event, and here the sensitivities depend on which DOAC is being taken, as well as on which assay methodology

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493Blood Transfus 2017; 15: 491-4 DOI 10.2450/2017.0301-16

Laboratory interference of DOAC

is being used. For example, in terms of congenital thrombophilia markers, chromogenic assays for protein C and antigenic assays for protein S are generally unaffected by the presence of DOAC10,11. However, clot-based assays for both protein C and protein S may be affected by any of the DOAC. For antithrombin, methods based on anti-FIIa inhibition are affected by dabigatran, whereas those based on anti-FXa inhibition will be affected by rivaroxaban and apixaban.

Other investigations commonly performed in patients who have had thrombotic event include a search for the acquired thrombophilia marker called the lupus anticoagulant, which is most commonly investigated using the APTT, the silica clotting time and/or dilute Russell viper venom time, all of which can be variably affected by DOAC10,11. As might be deduced from the above, APTT-based methods for the detection of lupus anticoagulant may be sensitive to dabigatran, in particular. Importantly, the dilute Russell viper venom time assay, unarguably the most important test currently used for detecting lupus anticoagulant, is quite strongly influenced by all the DOAC, leading to the solid possibility of both false-positive and false-negative diagnoses of lupus anticoagulant, depending on the patient tested and the DOAC influencing the test result10,11.

Among the effects of DOAC, the influence that these drugs have on activated protein C resistance (APCr) is somewhat under-evaluated. APCr is another marker of thrombophilia, both as potentially associated with congenital thrombophilia, usually in association with the factor V Leiden (FVL) mutation, or with acquired thrombophilia, usually as associated with elevated factor VIII levels in plasma. Certainly, previous studies evaluating APTT-based assays have noted a potential effect on APCr test results in the presence of DOAC10-13. Accordingly, the study by Gessoni et al.14, in the current issue of Blood Transfusion, adds yet another significant piece of information to the jigsaw of DOAC interference related to APCr testing. In this particular study, the authors investigated a prothrombinase-based APCr assay and found substantial interference of dabigatran, such that APCr ratios increased substantially in patients treated with dabigatran (ex vivo study), as well as in samples spiked with dabigatran (in vitro study). These effects were observed at very low concentrations of dabigatran, meaning that even trace amounts of the drug could influence test results. Most importantly, dabigatran at "therapeutic levels" could be shown to generate APCr ratios that would mask the presence of APCr and FVL (i.e., false-negative test). Given the known poor use that general clinicians normally make of haemostasis tests (APCr, FVL and other thrombophilia tests)15-19, with patients being either poorly selected by lack of pre-test

probability of familial thrombophilia, or assessed while on anticoagulant therapy instead of off therapy, together with the low relative incidence of such disorders in the general public, it can be hypothesised that the rising risk of identifying a false-negative APCr/FVL now probably exceeds the rate of true-positive APCr/FVL detection. In other words, testing patients for APCr while on DOAC therapy may mean that more individuals with FVL will be missed than will be patients with FVL identified when not on DOAC therapy.

In conclusion, we and others continue to advise against testing patients for thrombophilia markers, including APCr, while the patients are on anticoagulant therapy, of which dabigatran is now just one of many such anticoagulants affecting haemostasis testing. The risk of falsely diagnosing, or alternatively falsely excluding, a hereditary thrombophilia, such as APCr/FVL, or protein C, protein S or antithrombin, most likely exceeds, by several orders of magnitude, the likelihood of detecting true cases of hereditary thrombophilia, as previously reported during the era of vitamin K antagonists and heparinoids15-19, but now further accentuated by the existence and use of the DOAC4,5,9. In support of suggestions to avoid testing for these markers in patients being treated with anticoagulants, it should also be noted that only in very few cases (if any), would the identification of a prothrombotic risk factor significantly affect the early clinical management of patients with thrombosis; instead, a false-positive test result may trigger seriously inappropriate and potentially harmful treatments (e.g., immunosuppressive therapy in patients with false-positive results of clotting factor inhibitors such as lupus anticoagulant). In other words, in such cases "sometimes less is more".

The Authors declare no conflicts of interest.

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of Chest Physicians Antithrombotic Therapy and Prevention of Thrombosis Panel. Executive summary: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012; 141 (2 Suppl): 7S-47S.

2) Lippi G, Franchini M, Favaloro EJ. Pharmacogenetics of vitamin K antagonists: useful or hype? Clin Chem Lab Med 2009; 47: 503-15.

3) Favaloro EJ, Lippi G, Koutts J. Laboratory testing of anticoagulants - the present and the future. Pathology 2011; 43: 682-92.

4) Lippi G, Favaloro EJ. Recent guidelines and recommendations for laboratory assessment of the direct oral anticoagulants (DOACs): is there consensus? Clin Chem Lab Med 2015; 53: 185-97.

5) Favaloro EJ, Lippi G. Laboratory testing in the era of direct or non-vitamin K antagonist oral anticoagulants: a practical guide to measuring their activity and avoiding diagnostic errors. Semin Thromb Hemost 2015; 41: 208-27.

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6) Ruff CT, Giugliano RP, Braunwald E, et al. Comparison of the efficacy and safety of new oral anticoagulants with warfarin in patients with atrial fibrillation: a meta-analysis of randomised trials. Lancet 2014; 383: 955-62.

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8) Zolfaghari S, Harenberg J, Froelich L, et al. Development of recommendations to continue anticoagulation with one of the two types of oral anticoagulants based on the identification of patients' preference. Semin Thromb Hemost 2015; 41: 166-77.

9) Lippi G, Mattiuzzi C, Favaloro EJ. Thrombophilia testing in patients taking direct oral anticoagulants. Handle with care. Diagnosis 2014; 1: 311-2

10) Bonar R, Favaloro EJ, Mohammed S, et al. The effect of dabigatran on haemostasis tests: a comprehensive assessment using in-vitro and ex-vivo samples. Pathology 2015; 47: 355-64.

11) Bonar R, Favaloro EJ, Mohammed S, et al. The effect of the direct factor Xa inhibitors apixaban and rivaroxaban on haemostasis tests: a comprehensive assessment using in vitro and ex vivo samples. Pathology 2016; 48: 60-71.

12) Halbmayer WM, Weigel G, Quehenberger P, et al. Interference of the new oral anticoagulant dabigatran with frequently used coagulation tests. Clin Chem Lab Med 2012; 50: 1601-5.

13) Adcock DM, Gosselin R, Kitchen S, Dwyre DM. The effect of dabigatran on select specialty coagulation assays. Am J Clin Pathol 2013; 139: 102-9.

14) Gessoni G, Valverde S, Valle L, et al. Effect of dabigatran on a prothrombinase-based assay for detecting activated protein C resistance: an ex vivo and in vitro study in normal subjects and factor V Leiden carriers. Blood Transfus 2017; 15: 563-8.

15) Favaloro EJ, McDonald D, Lippi G. Laboratory evaluation of thrombophilia: the good, the bad and the ugly. Semin Thromb Hemost 2009; 35: 695-710.

16) Favaloro EJ, Mohammed S, Pati N, et al. A clinical audit of congenital thrombophilia investigation in tertiary practice. Pathology 2011; 43: 266-72.

17) Favaloro EJ, Reben R, Mohammed S, Koutts J. A clinical audit of antiphospholipid antibody testing in tertiary practice. Towards improved relevance in thrombophilia investigations? Intern Med J 2012; 42: 427-34.

18) Favaloro EJ. The futility of thrombophilia testing. Clin Chem Lab Med 2014; 52: 499-503.

19) Favaloro EJ, McDonald D. Futility of testing for factor V Leiden. Blood Transfus 2012; 10: 260-3.

Correspondence: Emmanuel J. FavaloroDepartment of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR)Westmead HospitalHawkesbury RoadWestmead, NSW, 2145, Australiae-mail: emmanuel.favaloro@health.nsw.gov.au

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