Measurement of non‐Coumarin anticoagulants and their effects on tests of Haemostasis: Guidance from the British Committee for Standards in Haematology

Abstract

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. The sections on heparin and low molecular weight heparin (LMWH) represent an update of the previously issued guidance (Baglin et al, 2006). 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 1. APTT Anti-Xa, Anti-IIa 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 inhibition (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 2, as knowing 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. 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-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). 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 3. 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) <1·5, although it should be noted that the International Sensitivity Index (ISI)/INR system, derived from patients receiving vitamin K antagonists, is not valid for patients receiving other anticoagulants. The PT was normal in 29% of samples collected 2–3 h post-dose during 150 mg twice daily dabigatran therapy (Hawes et al, 2013). Owren's PT methods, including Thrombotest, utilize a higher dilution of test plasma with lower potential for interference compared to other PT methods. There are case reports of marked elevation of the INR in the presence of dabigatran when the INR was determined using an unnamed Point-of-Care (POC) device (DeRemer et al, 2011) or the Hemochron Jr Signature device (Baruch & Sherman, 2011). The Hemochron Jr Elite POC analyser was more sensitive to the effect of dabigatran than five conventional laboratory PT methods in an ex vivo study (Hawes et al, 2013). The effect of DTIs, such as dabigatran, on tests where thrombin is directly added to plasma, including thrombin time and Clauss fibrinogen assay, depends on the concentration of thrombin in reagents and any pre-dilution of the plasma. Conventional thrombin times are very sensitive to the presence of dabigatran with greater than 10-fold prolongation at peak levels (Van Ryn et al, 2010), which do not return to normal at trough levels. In another in vitro study, plasma used in thrombin times became unclottable at dabigatran concentrations between 0·025 mg/l and 0·15 mg/l depending on the method used (Dager et al, 2012). For methods studied so far, a normal thrombin time can be used to exclude the presence of clinically relevant levels of dabigatran. Clauss fibrinogen assays without pre-dilution of plasma prior to analysis (Lindahl et al, 2011; Dager et al, 2012) underestimate fibrinogen concentrations. Methods which utilize higher thrombin concentrations and/or higher pre-dilution of plasma (typically 1 in 10 dilution or higher) are largely unaffected by dabigatran at concentrations up to 0·5 mg/l (Dager et al, 2012) or 1·0 mg/l (Lindahl et al, 2011; Douxfils et al, 2012). The effects of dabigatran on routine and specialist tests of haemostasis are summarized in Tables 4 and 5, respectively. Freyburger et al (2011) Lindahl et al (2011) Dager et al (2012) Baruch and Sherman (2011) Harenberg et al (2012) Douxfils et al (2012) Lindahl et al (2011) Douxfils et al (2012) Lindahl et al (2011) Van Ryn et al (2010) Lindahl et al (2011) Dager et al (2012) 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) Halbmayer et al (2012) Martinuzzo et al (2013) High concentrations of dabigatran can cause non-parallelism in factor assays and the underestimation of clotting factor levels is reduced at higher test plasma dilutions, although the effect of dabigatran may still occur at higher dilutions (Adcock et al, 2013). Additional higher test plasma dilutions should be analysed (see Mackie et al, 2013, for a full discussion of assay design in the presence of inhibitors). Thrombophilia testing is best avoided during therapy with DTIs, but if activated protein C resistance (APCR) testing is being performed to exclude FV Leiden, genetic testing is preferred because it would not be affected by the presence of DTIs. Factor Xa-based assays of antithrombin are preferred over thrombin-based assays unless there is evidence that a particular assay is unaffected by the level of anticoagulant in the sample. Chromogenic or antigen assays are preferable to clot-based assays of protein C and protein S in the presence of DTIs. Where argatroban is given for treatment of heparin-induced thrombocytopenia (HIT) the American College of Chest Physicians (ACCP) evidence-based clinical practice guidelines (Linkins et al, 2012) recommend that the dose is adjusted to maintain an APTT ratio of 1·5–3 times patient baseline APTT. The BCSH HIT guidelines (Watson et al, 2012) recommend an APTT ratio of 1·5–3·0 (but not exceeding 100 s). Reagents used for the determination of APTT may vary in their sensitivity to DTIs (Hirsh et al, 2008). Argatroban increases the INR, which complicates the transition from argatroban to vitamin K antagonists (VKA), and the BCSH recommends that patients on argatroban undergoing transition to warfarin should have an INR of ≥4·0 for 2 d prior to discontinuing argatroban (Watson et al, 2012). A number of studies have assessed the effects of argatroban on coagulation tests (Gosselin et al, 2004; Siegmund et al, 2008; Ivandic & 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 has been recommended as an alternative to heparin in patients with previous HIT who are HIT antibody-positive 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 6. The APTT was more sensitive than the PT and exhibited a non-linear 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. 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 anti-Xa 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 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). Expected plasma concentrations of rivaroxaban and apixaban are shown in Table 3. 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; et al, 2010; Samama et al, 2010). The effect of rivaroxaban on the APTT and PT is more than that of apixaban (Barrett et al, 2010). to the sensitivity of PT and APTT reagents to direct FXa inhibitors, the results are 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 prolongation of the PT is less marked with Owren's PT reagents which plasma and any drug is than with methods. Some ex vivo samples obtained from patients receiving rivaroxaban samples a normal PT using therapeutic rivaroxaban concentrations in one study (Mueck et al, 2011) but were prolonged at therapeutic levels in another et al, 2013). This reagent higher sensitivity to rivaroxaban in studies compared to most other et al, 2010; et al, 2011; Douxfils et al, 2012). A normal PT with and S reagents has been reported in the presence of up to rivaroxaban in samples from a patient with baseline PT to the lower limit of the normal range et al, 2013). times with were normal in all 10 ex vivo samples with mg/l rivaroxaban et al, 2013). More data are on ex vivo samples but a baseline PT may be to of PT results during therapy. studies have indicated that it may be to a rivaroxaban sensitivity to the specific in a system to and the of the results obtained with different reagents et al, et al, 2011) but the use of such an is not currently The effects of rivaroxaban on routine tests of haemostasis are shown in Table Samama et al (2010) et al (2011) Samama et al (2010) et al (2011) APTT ratio 5 Samama et al (2010) et al (2011) et al (2012) et al (2012) et al (2011) et al (2012) et al (2011) available PiCT and assay require to the sensitivity to measure the trough and peak levels of These clot-based assays are not specific and can be by other such as factor or The effects of rivaroxaban on specialist tests of haemostasis are shown in Table Samama and (2011) et al (2011) et al (2013) Martinuzzo et al (2013) ratios in one assay effect in another et al (2011) et al (2011) The effects of apixaban on a range of routine and specific coagulation assays have been assessed using apixaban from commercially available and pooled normal plasma (Douxfils et al, 2013). The effects on the PT were on the reagent of the PT was at concentrations of mg/l for of reagents The other reagent was more sensitive to the presence of There was some prolongation of the APTT in the presence of mg/l apixaban for 5 reagents There was a concentration response relationship for APTT and INR method in ex vivo samples but the sensitivity and the of tests as clinical of apixaban pharmacodynamic response (Frost et al, 2013). In a study, apixaban at up to mg/l relevant effect on Clauss some effect on the assay of factors and X and and was associated with interference in the Xa-based antithrombin assay, clot-based protein S assay and assays of factors and (Douxfils et al, 2013). monitoring of use of LMWH and is usually required (Baglin et al, 2006; et al, 2012). For treatment of monitoring for groups of such as the very severe renal and may provide These recommendations are by clinical trial data and laboratory results et al, et al, Samama & et al, et al, et al, et al, et al, The of the guidelines recommend that in or receiving therapeutic LMWH once or twice daily the drug should be to a anti-Xa of iu/ml in a sample h or iu/ml in a sample h after et al, 2012). The mechanism of action of LMWH is the of the of antithrombin to inhibit coagulation factor with the being factor The anti-IIa activity of LMWH is lower than its anti-Xa activity and is from than anti-Xa activity et al, is a and the anti-Xa activity of anti-IIa assay is therefore not a method of choice for measurement of LMWH and in clinical plasma the APTT is not a suitable method as it is largely to two anticoagulants. The PiCT has been by a number of groups for measurement of LMWH and activity et al, 2008; et al, 2008). PiCT is sensitive to it can be by other inhibitors and is therefore not specific for anti-Xa assay is the method of choice for monitoring There are many commercially available anti-Xa assays, some with exogenous which measure heparin and on the antithrombin in the patient plasma, which therefore measure anticoagulant should the method to ensure that the standard curve the concentration range One in commercial is the use of that provide accurate estimation of a range of anticoagulants. However, the sensitivity of anticoagulants such as FXa inhibitors and direct FXa inhibitors in anti-Xa assays are one calibrator accurate of concentration for all anticoagulants. added to plasma in vitro at concentrations of and 2 mg/l, was shown to prolong the APTT on and et al, 2006). reagents were In other studies the APTT was unaffected et al, and was not prolonged by anti-Xa activity (Linkins et al, The PT, as determined by many different methods, was prolonged by and Xa-based antithrombin assays were unaffected by at least mg/l et al, 2006). Peak levels of mg/l after mg and mg/l after mg have been reported et al, 2012). This guideline the relevant in the previous BCSH guideline on the use and monitoring of heparin (Baglin et al, 2006). In to the other non-coumarin anticoagulants discussed UFH is in an to effect without bleeding However, there is evidence to that monitoring of UFH therapy clinical outcomes et al, 2012). The test of choice for routine monitoring therapeutic doses of UFH has been the APTT in most There are a number of related to the use of the APTT. APTT methods vary in their to UFH et al, including variation between different of the same reagent in some et al, The used for additional variation et al, For local calibration of the APTT assay for each new number of reagent should be employed in the of the therapeutic range. In the study that established a therapeutic range for UFH monitoring using the APTT ratio et al, the range of which was associated with reduced risk of to a heparin level of iu/ml by or 0·3 to iu/ml by anti-Xa assay (Hirsh et al, 2008). The assay is not available or standardized so an anti-Xa assay should be used for This is by determination of the APTT ratio and anti-Xa on a of samples from subjects receiving UFH therapy and not on plasma The relationship is then used to the range of APTT ratios to 0·3 to though it should be noted that the evidence plasma heparin levels to the of bleeding or thrombosis is of low et al, 2012). The correlation between heparin level and APTT is as a of the lack of specificity of the APTT for a heparin effect, and the number of samples required for a valid assessment is not and samples have been used in some studies et al, & have may be required Some studies have indicated that monitoring of therapeutic UFH in the treatment of may not always be heparin was to be as safe and effective as LMWH in a randomized trial of patients with that APTT monitoring of heparin may not be et al, 2006). The of the guidelines for with treated with dosing should be used without monitoring than fixed or dosing with monitoring et al, 2012). A study of UFH anticoagulation intensity as of in patients concluded that an of time with an APTT prolonged to the level to iu/ml anti-Xa activity was associated with in and that patients receiving a heparin dose of at least a of of therapy time with this of APTT prolongation et al, 2011). concluded that if their were by clinical routine monitoring and heparin dose may be for patients receiving doses of at least For calibration or treatment monitoring, samples should be collected h after or dose (reviewed by 1995). are used should have a of in the after and should be within h of and within 4 h to the of heparin over time in et al, the sample can be collected which the sample from of heparin factor 4 et al, et al, is more rapidly in that a addition of as a of and of an effect that is if is used et al, The APTT may be within the therapeutic range therapy where factors other than a heparin effect to the prolongation of the APTT. patients should have a baseline APTT performed of therapy. the APTT is then the APTT method used is for the of monitoring. Where this baseline prolongation is by an reagent which the baseline APTT is can be The anti-Xa assay has a number of over the APTT for monitoring levels of and other clotting factors and the presence of anticoagulant do not chromogenic anti-Xa assays, which are therefore more specific for the heparin There is also evidence that the variation in results obtained with different Xa methods is less than that associated with different APTT including one study in which the anti-Xa activity in samples from patients receiving UFH from to for 8 different methods et al, there is evidence that anti-Xa assays can be used for monitoring UFH et al, when this be the test of choice for monitoring of the writing group the writing group if any new evidence available that would the of the recommendations in this or it The be and from the BCSH guidelines if it new recommendations are an be published on the BCSH guidelines are required to in level of evidence or additional evidence a new of the guidance be issued on the BCSH the and in this guidance is to be and accurate at the time of to the the BCSH the any for the of this