Measurement of DOAC and their effects on tests of haemostasis

guideline

Measurement of non-coumarin anticoagulants and their effects on tests of Haemostasis: Guidance from the British Committee for Standards in Haematology Steve Kitchen,1 Elaine Gray,2 Ian Mackie,3 Trevor Baglin4 and Mike Makris1,5 on behalf of the BCSH committee 1

Sheffield Haemophilia and Thrombosis Centre, Sheffield Teaching Hospitals NHS Trust, Sheffield, 2Haemostasis section, Biotherapeutics Group, National Institute for Biological Standards and Control, Potters Bar, 3Haemostasis Research Unit, Department of Haematology, University College London, London, 4Department of Haematology, Addenbrooke’s Hospital, Cambridge, and 5Department of Cardiovascular Science, University of Sheffield, Sheffield, UK

Keywords: Monitoring/measuring anticoagulation, Xa inhibitors, IIa inhibitors, heparin, low molecular weight heparin. The guideline group was selected to include UK-based medical, scientific and laboratory representatives. Publications known to the writing group were supplemented with additional papers identified by searching MEDLINE/Pubmed using the keywords direct thrombin inhibitors (DTI), direct Xa inhibitors, apixaban, argatroban, bivalirudin, dabigatran, fondaparinux, rivaroxaban, in combination with measurement, monitoring, coagulation assays, haemostasis assays and laboratory tests. The writing group produced the draft guideline, which was subsequently revised by consensus by members of the Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology (BCSH). The BCSH GRADE system was not applied to this guideline as it is inappropriate for laboratory studies. The guideline was then reviewed by a sounding board of c. 50 UK haematologists, the BCSH and British Society for Haematology (BSH) Committee and comments were incorporated where appropriate. The objective of this guideline is to provide healthcare professionals with clear guidance on the clinically important issues regarding the laboratory assessment of currently used non-coumarin anticoagulants and their impact on laboratory tests of haemostasis. A short summary of the effects of rivaroxaban and dabigatran on routine coagulation screens and assessment of anticoagulation intensity on behalf of the BCSH (Baglin et al, 2012) and international recommendations related to measurement of oral direct inhibitors (Baglin et al, 2013) have recently been published.

Correspondence: BCSH Secretary, British Society for Haematology, 100 White Lion Street, London N1 9PF, UK. E-mail: [email protected]

First published online 14 June 2014 doi: 10.1111/bjh.12975

The sections on heparin and low molecular weight heparin (LMWH) represent an update of the previously issued guidance (Baglin et al, 2006).

Anticoagulants in use in the UK The most common anticoagulants in use in hospitals in the UK are vitamin K antagonists, of which warfarin predominates. Recent BCSH guidelines have addressed warfarin management (Keeling et al, 2011) and this is not discussed in this document. Non-coumarin anticoagulants licensed for use in the UK at the time of writing are listed in Table I. Drug monitoring aims to use laboratory testing to optimize dosing to increase efficacy and/or safety. Monitoring of anticoagulants, other than warfarin, is primarily indicated for the intravenously administered drugs, such as unfractionated heparin (UFH), danaparoid, argatroban and bivalirudin. Monitoring is not required when anticoagulants are used for prophylaxis, where the anticoagulant effect is predictable and the drugs can be administered at a fixed weight-based dose. The efficacy of this approach has been established by the experience with the subcutaneously administered low molecular dose heparin (LMWH) and fondaparinux. More recently, the oral anticoagulants dabigatran, rivaroxaban and apixaban have been introduced without the intention of routine monitoring. These drugs have been shown in randomized trials to be effective and safe without monitoring (Connolly et al, 2009; Schulman et al, 2009; EINSTEIN Investigators, 2010; Patel et al, 2011). Arguments for and against laboratory monitoring of the new anticoagulants have been published (Bounameux & Reber, 2010; Mismetti & Laporte, 2010). The lack of a need for monitoring is based on the assumed similarity in pharmacokinetic and pharmacodynamic responses between individuals within a relatively wide therapeutic window. It has been estimated that the same dose of direct inhibitors of thrombin and activated factor X (Xa) can have up to 30% difference in thrombin generation inhibiª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 166, 830–841

Guideline Table I. Anticoagulants (excluding vitamin K antagonists) currently licensed for use in the UK.

Mode of administration

Drug

Oral

Dabigatran

Rivaroxaban Apixaban Intravenous or subcutaneous

Unfractionated heparin Danaparoid

Intravenous

Argatroban

Bivalirudin Subcutaneous*

Low molecular weight heparin*

Fondaparinux

Major mode of action Inhibition of FIIa

Inhibition of FXa Inhibition of FXa Inhibition of FIIa and FXa Inhibition of FXa Inhibition of FIIa Inhibition of FIIa Inhibition of FXa and, to a lesser degree, FIIa Inhibition of FXa

Potential laboratory tests Anti-IIa, Thrombin Clotting Time, ECT Anti-Xa, Anti-Xa, APTT Anti-Xa, Anti-IIa Anti-Xa APTT, ACT, ECT, Anti-IIa APTT, ACT, ECT, Anti-IIa Anti-Xa

Anti-Xa

Table II. Circumstances when measurement of anticoagulant concentration may be useful.

• • • • • • • • • • • • •

In the presence of spontaneous or traumatic haemorrhage Following suspected overdose When patients are taking another interacting drug To monitor efficacy in patients presenting with new thrombosis whilst on the anticoagulant When emergency surgery is required In patients due to have neuraxial anesthesia for elective or emergency procedures or surgery In patients requiring elective surgery and in whom the drug may still be present In patients with renal impairment When bridging from one anticoagulant to another To assess compliance At the extremes of body weight In subjects with prior intestinal surgery where it is unclear if absorption will be affected Trough levels may be useful to assess potential accumulation in very elderly patients

the concentration of the active drug or its effect may facilitate clinical management decisions. Some published data on the effects of drugs on laboratory tests of haemostasis are based on studies where drug is added to plasma/blood in vitro and any conclusions related to this type of sample are safer if also validated by analysis of samples from patients taking the drug.

ACT, Activated clotting time; APTT, activated partial thromboplastin time; ECT, Ecarin Clotting time. *Enoxaparin is also licensed for intravenous use in acute coronary syndrome.

Direct Thrombin Inhibitors (DTIs)

tion (Al Dieri & Hemker, 2010). Al Dieri and Hemker (2010) also speculated that bleeding would be more likely in high responders and thrombosis more likely in low responders. Clinical trials often exclude patients with impaired renal function, children, the very elderly, those with an increased bleeding risk and those at the extremes of body weight. The lack of a need to monitor demonstrated in the trials may therefore not be applicable to groups excluded from trial entry and the questioning of the extrapolation of overall trial benefit to the individual is not new (Rothwell, 1995). More interest may be focussed on laboratory measurements following an analysis of the association between plasma concentrations and efficacy and safety outcomes, which demonstrated that the risk of ischemic events was inversely related to trough drug levels (Reilly et al, 2014). This study concluded that both ischaemic stroke and bleeding outcomes were correlated with dabigatran plasma concentrations (Reilly et al, 2014). Despite the fact that monitoring is not required for many anticoagulants, it is important that clinical laboratories have the capacity to rapidly measure the concentration of all anticoagulants in the circumstances listed in Table II, as knowing

Specific chromogenic anti-IIa assays are available for the measurement of drugs that inhibit thrombin, including DTIs (Shepherd et al, 2011; Harenberg et al, 2012). These assays should be calibrated with product-specific calibrators which are available commercially for dabigatran and argatroban. For recommendations and general discussion of chromogenic assay design, see the BCSH guideline (Mackie et al, 2013). Thrombin-based clotting assays are available for the assay of dabigatran or argatroban. Product-specific calibrators should be used in all cases. One commercial assay, which uses a modified thrombin time, has been successfully used and shows a linear relationship between dabigatran concentration and clotting time up to at least 0
3 mg/l (Van Ryn et al, 2010; Douxfils et al, 2012; Harenberg et al, 2012) with a stated sensitivity of 0
008 mg/l (Douxfils et al, 2012). The test plasma is diluted (normally between 1 in 8 and 1 in 20) in normal plasma, rendering the assay largely independent of the test plasma fibrinogen concentration and function. This is a rapid assay that can be applied to most coagulometers, but the test is not fully specific for DTIs given that supra-

ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 166, 830–841

Measurement of dabigatran and argatroban concentrations

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Guideline therapeutic concentrations of UFH can prolong the clotting time. Another thrombin time method that uses diluted patient plasma also shows linearity between drug concentration and clotting time up to 0
5 mg/l, and is unaffected by heparin up to 1
0 iu/ml (Jones et al, 2012). Ecarin clotting time (ECT) assays can be used as a direct measure of DTIs. There is a correlation between ECT and plasma concentration of dabigatran up to 1
0 mg/l with ECT ratios of 2–4 after 150 mg twice daily dabigatran etexilate in healthy male subjects (Van Ryn et al, 2010), with steady state plasma concentrations of dabigatran occurring after 2–3 d and peak levels at 3 h post-dose. Data obtained from in vitro samples are also available (Douxfils et al, 2012; Harenberg et al, 2012). The ECT has not been standardized and can be affected by the concentration of prothrombin in plasma (reviewed by Samama & Guinet, 2011). Other tests which can be used for assay of DTIs include amidolytic ecarin assays (Guy et al, 2008; Siegmund et al, 2008; Douxfils et al, 2012) and semi-specific assays, such as Prothrombinase Induced Clotting Time (PiCT) (Fenyvesi et al, 2002; Guy et al, 2008; Douxfils et al, 2012). Liquid chromatography can be used to determine the concentration of dabigatran in plasma (Delavenne et al, 2012).

Recommendations  Dilute thrombin-based assays, ecarin-based assays or chromogenic anti-IIa assays (in the absence of heparin) are suitable for determination of plasma concentrations of dabigatran.  Assays to determine anticoagulant concentration should be calibrated with drug-specific calibrators.  Prothrombin time (PT) and activated partial thromboplastin time (APTT) should not be used to measure the plasma concentration of dabigatran.

Effects of dabigatran on tests of haemostasis Dabigatran etexilate is a pro-drug that is hydrolysed to its active form dabigatran in vivo, and in vitro studies therefore utilize this active form. Doses recommended for clinical use are 150 mg and 220 mg once daily, and 110 mg and 150 mg twice daily. Expected plasma concentrations are shown in Table III. The half-life is 12–17 h with normal renal function (Stangier et al,

2007; Wienen et al, 2007) and 18–27 h in the presence of moderate to severe renal impairment (Stangier et al, 2010). The APTT is usually prolonged by therapeutic doses of dabigatran, even at trough level, and the variation between results obtained with different reagents is relatively low (Van Ryn et al, 2010). Dabigatran does not always increase the APTT above the upper limit of the normal range. In one study, 18% of APTTs were normal during 150 mg twice daily dabigatran therapy (Hawes et al, 2013) in samples collected 2–3 h after dosing. The dose-response curve relating dabigatran concentration and prolongation of APTT is curvilinear, flattening at higher concentrations (Lisenfeld et al, 2006; Van Ryn et al, 2010). This effect, coupled with the lack of specificity of APTT for the presence of drug, indicates that the APTT is unsuitable for quantification of drug. The effect of dabigatran on the prothrombin time (PT) is less marked than on the APTT. Peak levels are usually associated with an International Normalized Ratio (INR) 0
1 mg/l drug If using thrombin-based assay (Xa-based assays unaffected)

PC assay

Overestimation for clot-based assay

Freyburger et al (2011) Adcock et al (2013) Lindahl et al (2011) Adcock et al (2013) Lindahl et al (2011) Douxfils et al (2012) Adcock et al (2013) Adcock et al (2013)

PS assay FVIII Inhibitor ACT

Overestimation for clot-based assay False positive Bethesda >0
2 mg/l Normal at 0
2 mg/l False positive at 0
05 mg/l and in ex vivo samples

DRVVT

False normal results may be possible with clotbased assays. Chromogenic assays unaffected Free PS antigen unaffected Ex vivo samples. Method studiedHemochron ACT-LR Standard or Normalized ratios affected

Adcock et al (2013) Adcock et al (2013) Hawes et al (2013)

Halbmayer et al (2012) Martinuzzo et al (2013)

FII, factor II; FV, factor V; FVII, factor VII; FVIII, factor VIII; FIX, factor IX; FX, factor X; FXI, factor XI; APC, activated protein C; APCR, activated protein C resistance; AT, antithrombin; PC, protein C; PS, protein S; ACT, activated clotting time; ACT-LR, Low Range Activated Clotting Time; DRVVT, Dilute Russell viper venom time.

Zorn, 2011) and indicate that the presence of argatroban can lead to underestimation of the level of clotting factors. However, given the relatively short half-life of c. 50 min, its effect on the measurement of clotting factors may not be so relevant.

Bivalirudin: monitoring and effects on clotting tests Bivalirudin has been recommended as an alternative to heparin in patients with previous HIT who are HIT antibodypositive and who require urgent cardiac surgery, and has been used for percutaneous coronary intervention in the UK (Watson et al, 2012). The half life is c. 25 min and the drug is eliminated by proteolytic cleavage and renal excretion (Koster et al, 2007). The PT, APTT and thrombin time were prolonged following addition of bivalirudin to pooled normal plasma, including concentrations that might occur in clinical use, in a study using one type of reagent in each test (Curvers et al, 2012). The results are shown in Table VI. The APTT was more sensitive than the PT and exhibited a nonlinear dose response curve. Neither test is suitable to monitor therapy. The response to bivalirudin of two diluted thrombin time assays and clotting or chromogenic ecarin-based assays was linear up to at least 5 mg/l (Curvers et al, 2012). The activated clotting time (ACT) was successfully used to monitor therapy during cardiopulmonary bypass, in which anticoagulation was considered adequate if a 2
5-fold or greater prolongation of the baseline ACT was obtained using the ACT method in local use (multiple centres, ACT methods not stated) (Koster et al, 2007), though ACT results are likely to vary between different commercially available methods. 834

Table VI. Effect of Bivalirudin on tests of haemostasis with one type of reagent (data from Curvers et al, 2012). Bivalirudin Concentration Test

1
0 mg/l

3 mg/l

5 mg/l

PT ratio APTT ratio Thrombin time ratio

c. 1
2 c. 2
2 c. 3
0

c. 2
1 c. 3
2 c. 5
5

c. 3
0 c. 4
0 c. 8
0

PT, prothrombin time; APTT, activated partial thromboplastin time.

Direct factor Xa inhibitors Measurement of oral anti-Xa agents The rational choice of measurement methods for direct FXa inhibitors is an anti-Xa assay. A number of in vitro and ex vivo studies indicated that anti-Xa chromogenic assays are more specific and sensitive than routine clotting test-based assays (Barrett et al, 2010; Becker et al, 2010; Samama et al, 2010, 2012). In addition, there is a better correlation between plasma concentrations, as measured by liquid chromatography/mass spectrometry, and levels estimated by anti-Xa assays than that obtained using the PT (Barrett et al, 2010). Commercial antiXa amidolytic assays are mainly designed for measurement of anti-Xa activity of LMWHs and some may require modifications for use with peak and trough levels of direct Xa inhibitors in treated patient plasma. In some studies, the activity of the FXa inhibitors was calibrated against a LMWH reference standard and the results expressed in iu/ml. This is not recommended as the mechanism of action of direct FXa inhibitors and LMWH is different and results showing valid comparison ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 166, 830–841

Guideline with LMWH are not robust. Product-specific calibrators must be used for accurate estimation of plasma level expressed in mass concentration (e.g. mg/l). The different chromogenic assays also have different dynamic ranges for rivaroxaban and apixaban (Barrett et al, 2010; Samama et al, 2012). Multiple calibrator and test plasma dilutions should be employed to ensure the test sample responses are within the range of the calibration curve and also to allow for assessment of linearity and parallelism (see Mackie et al, 2013). One study has reported overestimation of rivaroxaban levels (compared to the high performance liquid chromatography assay) with an anti- Xa assay utilizing exogenous antithrombin and the authors recommended against its use (Mani et al, 2012). The same study demonstrated the need for different calibrations for samples with high and low levels for some anti-Xa assays (Mani et al, 2012).

Recommendation  Anti-Xa chromogenic assays should be used to determine plasma concentration of direct FXa inhibitors.  Product-specific calibrator should be used and results should be expressed in mass concentration.  LMWH reference standards should not be used as calibrators for direct FXa inhibitors.  PT and APTT should not be used to measure the plasma concentration of Xa inhibitors.

Effects of rivaroxaban and apixaban on tests of haemostasis Expected plasma concentrations of rivaroxaban and apixaban are shown in Table III. The half-life of rivaroxaban is 7–11 h (Mueck et al, 2008) and that of apixaban is c. 12 h, with steady state occurring after 2–3 d of therapy (Frost et al, 2013). Both rivaroxaban and apixaban prolong the clotting times of clot-based assays, such as APTT, PT, dilute PT, dilute Russell viper venom time (DRVVT), PiCT and Heptest (Harder et al, 2008; Wong et al, 2008; Barrett et al, 2010; Samama et al, 2010). The effect of rivaroxaban on the APTT and PT is more pronounced than that of apixaban (Barrett et al, 2010). Due to the differing sensitivity of PT and APTT reagents to direct FXa inhibitors, the results are highly variable. For most reagents the PT is more sensitive to direct FXa inhibitors than the APTT. It should be noted that therapeutic APTT ratios established for UFH and INR for warfarin should not be used as guidance for safety and efficacy of rivaroxaban and apixaban. Rivaroxaban-associated prolongation of the PT is less marked with Owren’s PT reagents (in which plasma and any drug therein is diluted) than with Quick’s methods. Some ex vivo samples obtained from patients receiving rivaroxaban samples had a normal PT using STA-Neoplastine CI Plus (Stago, Asnieres sur Siene, France) despite therapeutic rivaroxaban concentrations in one study (Mueck et al, 2011) but were ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 166, 830–841

prolonged at therapeutic levels in another (Patel et al, 2013). This reagent showed higher sensitivity to rivaroxaban in spiking studies compared to most other thromboplastins (Samama et al, 2010; Hillarp et al, 2011; Douxfils et al, 2012). A normal PT with Innovin (Siemens, Marburg, Germany) and Thromborel S reagents (Siemens) has been reported in the presence of up to 270 ng/ml rivaroxaban in samples from a patient with baseline PT close to the lower limit of the normal range (van Veen et al, 2013). Prothrombin times with Recombiplastin 2G were normal in all 10 ex vivo samples with