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Comparison of monitoring methods for lepirudin: Impact of warfarin and lupus anticoagulant
Thrombosis Research 125 (2010) 538–544
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Comparison of monitoring methods for lepirudin: Impact of warfarin and lupus anticoagulant Birgitta Salmela a, Lotta Joutsi-Korhonen b, Ellen Saarela b, Riitta Lassila a,b,⁎ a b
Coagulation Disorders, Division of Hematology, Department of Internal Medicine, Finland HUSLAB Laboratory Services, Helsinki University Central Hospital, Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 23 November 2009 Received in revised form 1 February 2010 Accepted 2 February 2010 Available online 24 February 2010 Keywords: Direct thrombin inhibitor Ecarin Hirudin Lupus anticoagulant Oral anticoagulant
a b s t r a c t Introduction: Appropriate monitoring methods are needed for lepirudin, a direct thrombin inhibitor, as activated partial thromboplastin time (APTT) may under- or overestimate lepirudin. We compared APTT with thrombin-specific methods, also in the presence of warfarin and lupus anticoagulant (LA). Materials and Methods: Lepirudin i.v. was assessed in five patients (35 samples) and in vitro spiked plasma pools: normal control and plasma containing warfarin and LA. Wide dose-responses to lepirudin (0–4.0 µg/ml) were studied with APTT (Actin FSL®), Ecarin Chromogenic Assay (ECA®), chromogenic Anti-Factor IIa (Anti-FIIa, Hirudin Activity Assay®), Prothrombinase-induced Clotting Time (PiCT®), and plasma diluted Thrombin Time (dTT). Results: APTT both under- and overestimated in vivo lepirudin doses according to ECA® and Anti-FIIa, which matched completely in various plasma pools at all lepirudin doses (r= 0.99). APTT and PiCT® underestimated high lepirudin concentrations in normal plasma, and in LA-positive plasma they were invalid. In all plasma pools, dTT (1:16) indicated lepirudin well up to 1.0 μg/ml. Conclusions: ECA® or Anti-FIIa are preferable for lepirudin monitoring, because neither warfarin nor LA, interfered with them, and they were the most precise methods even for supratherapeutic doses. PiCT® reflected co-inhibition of FIIa and FXa, but was disturbed, like APTT, by LA and high lepirudin. Further experience of laboratory monitoring is valuable in this era of new anticoagulants. © 2010 Elsevier Ltd. All rights reserved.
Introduction The recombinant hirudin lepirudin is a potent direct thrombin inhibitor (DTI) [1] which blocks both the active and the fibrinogen binding sites of free and clot-bound thrombin [2,3]. Lepirudin is indicated in heparin-induced thrombocytopenia (HIT) [4] but is also effective in prophylaxis and treatment of thrombosis [5–10]. An appropriate laboratory method for lepirudin monitoring is crucial due to its narrow therapeutic range and high potency. Activated Partial Thromboplastin Time (APTT) is used for monitoring heparin and lepirudin [11], but APTT has poor reproducibility, a non-linear doseresponse, with a plateau even at therapeutic levels, and an inability to detect either lepirudin overdoses or the high concentrations necessary in cardiopulmonary bypass surgery (CPB) [7,12–14].
Abbreviations: APTT, activated partial thromboplastin time; LA, lupus anticoagulant; ECA®, ecarin chromogenic assay; Anti-FIIa, chromogenic Anti-Factor IIa assay; PiCT®, prothrombinase-induced clotting time; dTT, plasma diluted thrombin time; DTI, direct thrombin inhibitor; HIT, heparin-induced thrombocytopenia; CPB, cardiopulmonary bypass surgery; INR, international normalized ratio; AT, antithrombin. ⁎ Corresponding author. Coagulation Disorders, Division of Hematology, Department of Internal Medicine, Helsinki University Central Hospital, P.O. Box 340, 00029 HUS, Helsinki, Finland. Tel.: + 358 40 517 5547; fax: +358 9 471 74504. E-mail address: riitta.lassila@hus.fi (R. Lassila). 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.02.002
Specific direct prothrombin activation methods have been developed for DTI monitoring: ecarin-based methods, e.g. Ecarin Clotting Time (ECT) [15] and Ecarin Chromogenic Assay (ECA®) [16] and Prothrombinase-induced Clotting Time (PiCT®) [17]. Another quantitative measurement, additional to ECA®, is a thrombin-based chromogenic, Anti-Factor IIa (Anti-FIIa) assay [18]. Plasma diluted Thrombin Time (dTT) has been modified from Thrombin Time for lepirudin monitoring [19], though parallel studies of these methods are limited [11,20]. According to our understanding, APTT, ECA®, PiCT®, Anti-FIIa, and dTT have not been compared. The challenges that clinicians face in using APTT are highlighted by our analysis of patient plasma samples obtained during lepirudin treatment. We compared the different methods (APTT, ECA®, PiCT®, Anti-FIIa, and dTT) to assess the intensity of anticoagulation in patient samples and their performance in vitro in assessing lepirudin doseresponses, especially in the presence of warfarin and lupus anticoagulant (LA). Materials and Methods Patient samples Lepirudin has been used at our University Hospital in approximately 50 patients with HIT-related or heparin-resistant thrombosis
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[8–10]. We analyzed 35 plasma samples from five random patients on i.v. lepirudin (Refludan®; Pharmion, Cambridge, UK) (Table 1). Their thrombosis had been treated with unfractionated or low-molecularweight heparin before the switch to lepirudin. Doses of lepirudin had been adjusted on the basis of APTT [21] and of dTT, when available. APTT target ratios were 1.5 to 2.5 depending on the clinical situation. All patients had normal creatinine levels. The study was approved by the hospital's Institutional Review Board and Ethics Committee. Blood samples were collected by antecubital venipuncture into vacuum tubes containing 3.2% sodium citrate (109 mM). Thereafter, plasma was prepared by centrifugation (2,000 ×g, 10 min). Only the upper or middle third of the plasma was collected. After routine laboratory measurements the plasma was stored at -70 °C for less than 12 months. Lepirudin spiking in vitro Plasma was spiked to achieve a wide concentration range of lepirudin (0–4.0 μg/ml). Three different types of pooled plasma were used: 1) Standard Human Plasma (Siemens Healthcare Diagnostics, Marburg, Germany) as normal control plasma containing the standard range of coagulation factors. 2) Commercial warfarin plasmas (Coumadin® plasma, CliniSys Associates, Ltd., USA) at three INR (International Normalized Ratio) levels: 1.5, 2.5, and 3.9 (corresponding to INRs of 1.5, 2.5, and 3.6 provided by CliniSys). 3) Three LA-positive plasma pools (LA1–LA3) were combined from 8 to 21 samples screened positive or strongly positive in two LA tests: the dilute Russell's Viper Venom Time (dRVVT) (DVVtest®, American Diagnostica Inc., Germany) and APTT test (IL Test APTTSP, Instrumentation Laboratory, Italy). Levels of cardiolipin and β2-glycoprotein I antibodies (Varelisa, Cardiolipin IgG Antibodies and Beta-2-Glycoprotein I IgG Antibodies, Phadia GmbH, Freiburg, Germany) were assessed (normal for both b 15 U/ml). In the LApositive plasma pools, antibody levels were 70 and N 100 U/ml (LA1), 40 and 30 U/ml (LA2), and 30 and 50 U/ml (LA3), respectively. The activities of FII (reference range 68–144%), FX (79–146%) (Coagulation Factor II and X Deficient Plasmas, Siemens), and antithrombin (AT; 84–108%) (Berichrom AT III, Siemens) were analyzed before and after addition of lepirudin (1.5 µg/ml) to the plasma pools. The samples were vortex-mixed and immediately studied. Table 1 Lepirudin indications and dosing in patients with proceeding heparin-resistant thrombosis. Patient Age Indication (yrs) 1#
24
2#
28
3#
79
4#
51
5#
38
Pulmonary embolism Hepatic venous thrombosis Critical leg ischemia (inoperable) Critical leg ischemia (inoperable)
Other diagnosis
Dilative cardiomyopathy Paroxysmal nocturnal hemoglobinuria DM, antiphospholipid antibodies DM, CAD, atrial fibrillation, renal transplant, FVIII:c† Critical leg ischemia Horton's neuralgia, (inoperable) with FVIII:c† aortic thrombosis
Median dosage (mg/kg/h) (range)* 0.2 (0.13-0.23) 0.07 (0.05-0.11) 0.06 (0.06-0.09) 0.04 (0.01-0.12)
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Monitoring methods APTT was measured by means of Actin® FSL reagent (Siemens) (reference range 23–33 s and analytic range 8–180 s). For patients #1 to #4, lepirudin therapies had been initially monitored with another APTT reagent (IL Test APTT-SP, reference range 24–40 s). Prothrombin Time (PT) (reference range 70–130%) and INR were studied by the Nycotest® PT reagent (Axis-Shield PoC AS, Oslo, Norway). As specific assays, we used four (see below) monitoring methods for lepirudin. All analyses were performed by a BCS® XP coagulation analyzer (Siemens). Calibrations and controls were performed according to manufacturer's instructions. Ecarin Chromogenic Assay (ECA®) Ecarin, a snake venom metalloprotease, degrades prothrombin and activation products mainly to meizothrombin. An ecarin-based chromogenic assay has earlier been developed for quantifying hirudin (at 405 nm) (HaemoSys® ECA®-H, JenAffin GmbH, Jena, Germany) [16,22]. ECA® prothrombin buffer (100 µl), substrate (25 µl) and the sample (25 µl) were gently mixed and incubated (37 °C, 1 min) and measured after adding ecarin reagent (50 µl). The analytical range was 0–2.0 µg/ml. ECA® is independent of prothrombin and fibrinogen. Both intra- and interassay variations (n = 20, CV%) at 1.5 µg/ml of lepirudin were 7%. Chromogenic Anti-FIIa Assay (Anti-FIIa) An Anti-FIIa assay (Siemens) was used to quantify lepirudin activity in plasma. This method is independent of antithrombin and fibrinogen levels [18]. Plasma sample (25 µl) and substrate (50 µl) were mixed, and the reaction was started with thrombin reagent (250 µl), which induces complex formation 1:1 with lepirudin. Residual thrombin activity was determined by kinetic testing of the increase in absorbance (1 min, at 405 nm). The analytical range was 0–2.3 µg/ml. Intra- and interassay (n = 20) variations were 3–4%. Prothrombinase-induced Clotting Time (PiCT®) The plasma-based functional PiCT® assay (Pefakit®, Pentapharm, Basel, Switzerland) measures anticoagulant activity by FXa or FIIa inhibition, or both [17]. In PiCT®, prothrombinase complex activates coagulation, and the activator reagent contains phospholipids, calcium, FX, and preactivated FV (activator: RVV-V from Russell's viper venom). A two-step coagulation test was performed: incubation (180 s, 37 °C) of plasma (50 µl) with the activator reagent (50 µl) and addition of the start reagent (50 µl 25 mM CaCl2). Clotting time was referred to the standard curve using Pefakit® PICT® Calibrator Hirudin plasmas. The reference range was 19–31 s, and the measurement range was 20–250 s (corresponding to 0–3.0 µg/ml of lepirudin) according to manufacturer's instructions. Intra- and interassay (n = 20) variations were 12%. Plasma Diluted Thrombin Time (dTT) The plasma dTT procedure was developed when TT exceeded 140 s (analytic range 12–140 s) and was too sensitive for therapeutic lepirudin concentrations. TT was performed according to manufacturer's instructions. Buffer solution (5 ml) was added to BC Thrombin reagent® (Siemens) (22 °C, 30 min), plasma (40 µl) was supplemented with BC Thrombin reagent® (100 µl), and clotting time registered up to 150 s. If no clotting was detected, the samples were further diluted with pooled normal plasma. We used serial dilutions from 1:2 to 1:16 until dTT was measurable, or until dilution 1:16.
0.11 (0.1-0.15)
* Lepirudin was started at 0.1–0.15 mg/kg/h without a bolus dose owing to prior anticoagulation therapy. † FVIII:c, factor VIII coagulant activity was high N 200% (normal range 52–148%). DM, diabetes mellitus; CAD, coronary artery disease.
Statistical analysis Statistical analysis was performed with SPSS® 17.01 software (SPSS Inc., Chicago, IL). Intraclass correlation coefficients (ICC; OneWay Random model, with 95% confidence intervals) were determined
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for ECA®, Anti-FIIa, and respective lepirudin concentrations by reliability analysis, whereas Spearman's correlation coefficient (r, 2tailed significance) was used for APTT, PiCT®, and dTT and respective lepirudin concentrations. Significance was set at p b 0.05. Results Monitoring of lepirudin in vivo Patient plasma samples obtained during lepirudin therapy were analyzed with APTT, ECA®, Anti-FIIa, PiCT®, and dTT for dose-responses and possible monitoring difficulties. Both elevated bleeding risk (patients #2 with anemia and thrombocytopenia and #4 with transient renal impairment) and the timing of possible surgery affected treatment strategies in these cases of heparin-resistant thrombosis (Table 1). No clinically significant bleeding occurred. Specific monitoring methods revealed important pitfalls of APTT and the clinical challenges in administering efficacious but safe lepirudin therapy. In an example of overdosing (Fig. 1, patient #1), APTT underestimated lepirudin dosage, whereas ECA®, PiCT®, and Anti-FIIa indicated evident overdosing. The patient was overweight (body mass index 33 kg m-2), and the recommended maximum dosage was nearly exceeded (Table 1). In a patient with paroxysmal nocturnal hemoglobinuria (PNH) and hepatic venous thrombosis (Fig. 1, patient #2), lepirudin dose was kept prophylactic rather than therapeutic, because of anemia and low platelet count. An LA-positive patient's (#3) lepirudin dosing was based on the dTT (1:8 dilution), as
the APTT method was oversensitive (Fig. 1). To avoid overdosing, the infusion was repetitively withheld unnecessarily according to APTT (Actin® FSL), while ECA® and Anti-FIIa indicated underdosing of lepirudin. APTT's insensitivity to dose escalation was seen when it was prolonged only by 5 s versus doubling in ECA® and Anti-FIIa tests (Fig. 1, patient #4). All methods indicated successful dosing in patient #5 (Fig. 1). PiCT®, like APTT, overestimated lepirudin doses in the patients with LA (#3), PNH (#2) and a renal transplant (#4), modestly accumulating lepirudin (Fig. 1). In addition to APTT, dTT (1:8 dilution) was used to guide dosing in patients #1 to #4, but this dilution seemed inadequate for therapeutic lepirudin. At 1:16 dilution, dTT supplemented APTT relatively well (data not shown). In all, inadequate lepirudin dosing, especially in the presence of LA, are evident in APTT responses in comparison with ECA® and Anti-FIIa. Monitoring of lepirudin in vitro Lepirudin concentrations in vitro were chosen to cover the prophylactic, therapeutic (ca. 0.6–1.0 μg/ml), and supratherapeutic levels (2.5–4.0 μg/ml) desired during CPB surgery. Moreover, the combined effects of lepirudin, warfarin-, and LA-positivity were assessed in respective plasma pools. INR and PT (%) The additive anticoagulant effect of lepirudin on INR and PT (%) was measured in three warfarin-containing plasma pools at various
Fig. 1. Monitoring lepirudin treatment in five patients by means of APTT, ECA®, PiCT®, and Anti-FIIa methods. Repetitive plasma samples (on x-axis sample numbers) were obtained during i.v. lepirudin therapy (3-8 days). APTT (s) and respective ECA®, PiCT®, and Anti-FIIa (μg/ml) results in separate panels. Therapeutic range for lepirudin indicated by horizontal lines. Examples of: Overdosing (patient #1): At APTT target, lepirudin concentrations were supratherapeutic according to ECA®, PiCT®, and Anti-FIIa. Underdosing (patient #2): Lepirudin was subtherapeutic according to all methods. Interference by lupus anticoagulant (LA) (patient #3): LA disturbed both APTT and PiCT®, while according to ECA® and AntiFIIa, lepirudin therapy was subtherapeutic. The poor sensitivity of APTT to dose escalation (patient #4): APTT seemed stable despite doubled lepirudin action according to ECA® and Anti-FIIa. Successful laboratory monitoring (patient #5): APTT remained at its target, and ECA® as well as Anti-FIIa indicated appropriate concentrations.
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INR levels (Fig. 2A). At the lowest, INR 1.5, the combined impact was modest even at the highest lepirudin concentration (4.0 μg/ml) (Fig. 2A), but at therapeutic INR levels, lepirudin markedly raised INR. In LA-positive plasma, PT (%) declined with increasing lepirudin concentrations (Fig. 2B), whereas LA had no effect on INRs. APTT Dose-response curves for APTT remained linear only at the lower therapeutic range of lepirudin. Thereafter, APTT reached a plateau in the normal, warfarin-, or LA-positive plasma pool (Fig. 3A, B). APTT was prolonged in all warfarin pools (Fig. 3A). The presence of LA immediately prolonged APTT, resulting in non-measurable clotting, even at low lepirudin concentrations (0.4–0.7 μg/ml) (Fig. 3B). APTT correlated modestly with lepirudin concentrations in different plasma pools (r = 0.69, p b 0.001). ECA® and Anti-FIIa ECA® responses linearly paralleled doses up to 3.0 µg/ml and agreed with spiked lepirudin throughout the dose range (Fig. 4A). Importantly, the presence of warfarin or LA did not disturb ECA® (ICC = 0.99, 95% CI 0.98–0.99, p b 0.001) (Fig. 4A). Anti-FIIa also linearly paralleled lepirudin responses in the various plasma pools. Again, no interference of warfarin or LA was observable (ICC = 0.99, 95% CI 0.985–0.995, p b 0.001) (Fig. 4B). Even the lowest lepirudin
Fig. 2. Responses of INR (A) and PT (B) to lepirudin in vitro in normal, warfarin-, and LApositive plasma pools. Three INR pools (1.5, 2.5, and 3.9) (A) and three LA pools (LA1– LA3) were used (B). Results in normal plasma serve as the reference. Lepirudin and warfarin had an additive effect to INR (A). PT decreased progressively with increasing lepirudin concentration in the presence of LA (B). *Normal refers to normal plasma (at baseline INR was 1.0, PT 96% and LA negative).
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concentrations (0.1 µg/ml) were adequately detected by ECA® and Anti-FIIa methods. These chromogenic methods produced in all plasma pools practically identical results (ICC = 0.99, 95% CI 0.989– 0.996, p b 0.001).
PiCT® PiCT® displayed a relatively linear dose-response to lepirudin in normal plasma in the therapeutic range (r = 0.83, p b 0.001), however, supratherapeutic levels were severely underestimated (Fig. 5A). Coexisting warfarin modified the PiCT® results, even at INR 1.5, precluding separate analysis of lepirudin's anticoagulant effect (Fig. 5A). The presence of LA prolonged PiCT®, like APTT, and PiCT® failed to detect lepirudin in all LA-positive plasma pools (Fig. 5B). Both warfarin and LA interfered with PiCT® (Fig. 5A, B).
dTT As dTT (1:8 dilution) remained below 140 s (i.e. maximum) only at low lepirudin concentrations (b0.7 µg/ml), a further dilution to 1:16 was necessary to extend the measurement range. Responses of dTT to lepirudin up to 1.0 µg/ml appeared relatively linear in normal, warfarin-, and LA-positive plasma pools (Fig. 6A, B). The sensitivity of dTT to increasing lepirudin doses was weaker than with ECA® and Anti-FIIa, but it improved when dTT sample was further diluted from 1:8 to 1:16 (r = 0.93 at 1:8, r = 0.97 at 1:16; both p b 0.001).
Fig. 3. Dose responses of lepirudin according to APTT in vitro in normal (A, B), warfarin(A), and LA-positive (B) plasma pools. Three INR pools (1.5, 2.5, and 3.9) and three LA pools (LA1–LA3) were used. Results in normal plasma served as reference values. APTT reached a plateau in normal plasma (A, B). Warfarin at INR 2.5 and 3.9 together with lepirudin prolonged APTT (A). LA extensively prolonged APTT (B).
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Fig. 4. Dose responses of lepirudin according to ECA® (A) and Anti-FIIa (B) in vitro in normal plasma, warfarin-, and LA-positive plasma pools. Three INR pools (1.5, 2.5, and 3.9) and three LA pools (LA1–LA3) were used. Neither warfarin nor LA interfered with the linear dose-responses of ECA® or Anti-FIIa.
Comparison of monitoring methods Normal control plasma APTT showed no linear dose-response at lepirudin concentrations above 0.5 μg/ml (Fig. 3A). In contrast, both ECA® and Anti-FIIa data were linear throughout the wide concentration range (Fig. 4). Importantly, ECA® and Anti-FIIa methods detected supratherapeutic lepirudin concentrations agreeing with the spiking, in contrast to PiCT®, which indicated 20% lower concentrations of lepirudin in the upper therapeutic range (Fig. 7). In addition, dTT (1:16 dilution) well assessed the dose-responses of lepirudin up to 1.0 μg/ml (Fig. 6A). FII, FX, and AT activities were normal with and without lepirudin (1.5 μg/ ml). Warfarin-containing plasma The co-effect of lepirudin and warfarin prolonged APTT without any significant difference at an INR between 2.5 and 3.9 (Fig. 3A). Both ECA® and Anti-FIIa methods detected lepirudin doses linearly regardless of distinct vitamin K-dependent coagulation factor deficiencies (Fig. 4). PiCT®, however, indicated higher levels, and was progressively distorted by warfarin at INR ≥1.5 and lepirudin above 0.7 μg/ml (Fig. 5A). An increasing warfarin concentration (INR) seemed not to affect dTT (1:16 dilution) results (Fig. 6A). As expected, FII activities were low, paralleling INR at baseline, and were further diminished after addition of lepirudin (1.5 μg/ml) (INR 1.5: from 53 to 37%, INR 2.5: from 28 to 10%, and INR 3.9: from 19 to 8%). After addition of lepirudin (1.5 μg/ml) the activities of FX remained at the same low levels (INR 1.5: from 41 to 36%, INR 2.5: 11% and INR 3.9: 9%), but AT remained normal (data not shown).
Fig. 5. Dose responses of lepirudin according to PiCT® in vitro in normal plasma (A, B), warfarin-, (A) and LA-positive (B) plasma pools. Three INR pools (1.5, 2.5, and 3.9) and three LA pools (LA1–LA3) were used. Results in normal plasma served as reference values. Coexisting warfarin clearly prolonged PiCT®, even at INR 1.5 (A). The presence of LA uniformly prolonged PiCT® (B).
LA-positive plasma Both APTT and PiCT® showed prolonged coagulation times in the presence of LA, and lepirudin could not be traced (Figs. 3B, 5B). In contrast, ECA® and Anti-FIIa predicted lepirudin doses in LA plasma (Fig. 4). The three LA-positive pools produced similar dTT data (Fig. 6B). Lepirudin (1.5 μg/ml) somewhat reduced FII activity (LA1: 65%, LA2: 63%) in LA-positive pools, whereas FX and AT activities remained normal. Discussion Currently, as a result of the lack of a reliable monitoring method, the antithrombotic potential of lepirudin may be underused regarding therapy and CPB surgery. According to our results both chromogenic methods were precise and appeared suitable for monitoring of lepirudin-induced anticoagulation, even including supratherapeutic levels. Importantly, ECA® and Anti-FIIa were reproducible and unaffected by warfarin or LA, common clinical confounding factors. In contrast, APTT underestimated lepirudin in the upper therapeutic range and appeared unsuitable in the presence of warfarin or LA. Unfortunately, PiCT® seemed to show some of the same limitations. Although ECA® and Anti-FIIa methods provide accurate options for lepirudin monitoring, a functional method could indicate residual inhibition of coagulation when bridging with other anticoagulants (i.e. heparin and warfarin). Additionally, lepirudin may produce distinct responses in coagulation assays during thrombosis and fibrinolysis. APTT is severely limited, as it may indicate falsely high lepirudin levels
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Fig. 6. Dose responses of lepirudin in vitro according to dTT (1:16 dilution) in normal plasma (A, B), warfarin- (A), and LA-positive (B) plasma pools. Three INR pools (1.5, 2.5, and 3.9) and three LA pools (LA1–LA3) were used. Results in normal plasma served as reference values. The presence of warfarin (A) and LA (B) had no effect on dTT. † Lowest lepirudin concentrations (0 and 0.1 µg/ml) are presented with undiluted TT, whereafter, TT became unmeasurable without further dilution.
under several clinical situations, including prothrombin deficiency, liver disease, disseminated intravascular coagulation, or the presence of LA, or vitamin K antagonists [7]. In the case of warfarin, our findings confirm the previous observations [23,24] in a wider range of INR (1.5-3.9) and analytical methods. For instance, in HIT, a premature disruption of lepirudin infusion due to falsely high APTT may be
Fig. 7. Comparison of dose responses of lepirudin by ECA®, Anti-FIIa, and PiCT® methods in normal plasma. ECA® and Anti-FIIa produced relative similar, exact, and linear dose responses throughout the wide concentration range of lepirudin (0-4.0 µg/ml). PiCT® underestimated lepirudin concentrations above 1.0 µg/ml.
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deleterious during thrombosis and possible “rebound” thrombin generation [7]. The risk that lepirudin tapering would be unnecessary could be avoided by use of assays like ECA® and Anti-FIIa, both independent of prothrombin or fibrinogen, and of the presence of LA and warfarin. PiCT underestimated lepirudin in the upper therapeutic range in normal plasma in comparison with ECA® and Anti-FIIa (Fig. 7), as noted earlier [13], the underlying reason remaining speculative. On the other hand, the enhanced warfarin effect on PiCT® could signal the presence of high lepirudin doses, when bridging oral anticoagulants and vice versa. In case of LA-positivity, however, PiCT®, like APTT, is invalid. Besides APTT, if ECA® or Anti-FIIa methods are unavailable, dTT could help in adjusting appropriate lepirudin dosing, as suggested also by others [19]. In the therapeutic range of lepirudin, dTT at 1:8 exceeds the measurement range (140 s), whereas dTT at 1:16 should remain under a specified limit (in our case ∼ 50 s), to be assessed by each laboratory. The anticoagulant effect of lepirudin is more predictable than that of heparin due to the lack of cofactors, to non-specific binding characteristics onto cellular surfaces, and to neutralization by proteins [25,26]. Lepirudin has a short plasma half-life (1.5–2 h), but on the clot surface and in the extravascular space it remains active far longer [3], making rebound-like action plausible. Coagulation activity is inhibited at low concentrations, whereas higher concentrations are needed to inhibit development of platelet-dependent arterial thrombosis [25,27]. Safe and effective treatment with lepirudin demands a reliable monitoring method to avoid under- and overdosing; especially during warfarin therapy or its initiation after lepirudin or in the presence of LA. Moreover, in association with impaired renal function the half-life of lepirudin may be prolonged, even up to 30fold [28,29]. Small but effective doses of lepirudin can be administered, according to ECA® or Anti-FIIa. In our patients, lepirudin dosing turned out to be less than intended (Table 1), but deliberate underdosing (patients #2 to #4) occurred due to the limitations of APTT. Indeed, recently published data and guidelines recommend lower than earlier lepirudin dosing levels [4,7,21,30–32]. The ultimate target is to avoid hemorrhagic problems, while achieving appropriate therapeutic inhibition of thrombin in pharmacodynamically vulnerable patients. Both ECA® and Anti-FIIa methods can be adapted for automated analyzers. Anti-FIIa reagents are stable and may be stored longer than ECA® reagents. ECA® is also feasible for point-of-care testing. Clinically, bedside methods would be useful, especially during CPB surgery. ECT is more widely used than ECA®, as it has been long available. ECT shows a linear association with lepirudin, even at a higher concentration than ECA® (up to 5.0 µg/ml) [15]. Both ECT and ECA® are independent of LA, warfarin, and fibrinogen, but ECA® overcomes the limitation of ECT during prothrombin deficiency [16,22]. The importance of comparative monitoring data on various DTIs and FXa inhibitors is increasing as many new anticoagulants, i.e. dabigatran and rivaroxaban, are emerging [33,34]. During overdosing, severe bleeding complication, emergency surgery, or under renal insufficiency, a drug-specific and reliable monitoring method augments appropriate management. Our study results apply to lepirudin only, and for other DTIs the dose-responses need to be individually studied. ECA®, however, seems to provide a linear dose-response also with other DTIs, i.e. argatroban and bivalirudin (according to manufacturer's instruction), in contrast to APTT, PiCT®, and ECT [13,22,35,36]. In conclusion, automated quantitative analysis of lepirudin by means of ECA® or Anti-FIIa assays can be recommended to improve safety in patients treated with the potent DTI lepirudin, even during CBP surgery. When lepirudin is administered with oral anticoagulants or heparins, PiCT® or APTT together with chromogenic ECA® or AntiFIIa methods may be helpful in assessing prevailing anticoagulant
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activity. Finally, ECA® or Anti-FIIa methods are appropriate for monitoring lepirudin under the influence of warfarin as well as LA. Conflict of interest statement None. Acknowledgements None. References [1] Harvey RP, Degryse E, Stefani L, Schamber F, Cazenave JP, Courtney M, et al. Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech, Hirudo medicinalis. Proc Natl Acad Sci U S A 1986;83:1084–8. [2] Fenton II JW, Villanueva GB, Ofosu FA, Maraganore JM. Thrombin inhibition by hirudin: how hirudin inhibits thrombin. Haemostasis 1991;21:27–31. [3] Agnelli G, Renga C, Weitz JI, Nenci GG, Hirsh J. Sustained antithrombotic activity of hirudin after its plasma clearance: comparison with heparin. Blood 1992;80: 960–5. [4] Warkentin TE, Greinacher A, Koster A, Lincoff AM, American College of Chest P. Treatment and prevention of heparin-induced thrombocytopenia: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest 2008;133:340S–80S. [5] Fischer KG. The role of recombinant hirudins in the management of thrombotic disorders. BioDrugs 2004;18:235–68. [6] Di Nisio M, Middeldorp S, Buller HR. Direct thrombin inhibitors. N Engl J Med 2005;353:1028–40. [7] Greinacher A, Warkentin TE. The direct thrombin inhibitor hirudin. Thromb Haemost 2008;99:819–29. [8] Kuitunen A, Suojaranta-Ylinen R, Raivio P, Kukkonen S, Lassila R. Heparin-induced thrombocytopenia following cardiac surgery is associated with poor outcome. J Cardiothorac Vasc Anesth 2007;21:18–22. [9] Salmela B, Nordin A, Vuoristo M, Mäkisalo H, Numminen K, Lassila R. Budd-Chiari syndrome in a young female with factor V Leiden mutation: successful treatment with lepirudin, a direct thrombin inhibitor. Thromb Res 2008;121:769–72. [10] Salmela B, Albäck A, Räike P, Lepäntalo M, Lassila R. A direct thrombin inhibitor, lepirudin, for thrombophilic patients with inoperable critical limb ischemia. Thromb Res 2009;123:719–23. [11] Gray E, Harenberg J, ISTH Control of Anticoagulation SSC Working Group on Thrombin Inhibitors. Collaborative study on monitoring methods to determine direct thrombin inhibitors lepirudin and argatroban. J Thromb Haemost 2005;3: 2096–7. [12] Nowak G. Clinical monitoring of hirudin and direct thrombin inhibitors. Semin Thromb Hemost 2001;27:537–41. [13] Fenyvesi T, Jorg I, Harenberg J. Monitoring of anticoagulant effects of direct thrombin inhibitors. Semin Thromb Hemost 2002;28:361–8. [14] Gosselin RC, Dager WE, King JH, Janatpour K, Mahackian K, Larkin EC, et al. Effect of direct thrombin inhibitors, bivalirudin, lepirudin, and argatroban, on prothrombin time and INR values. Am J Clin Pathol 2004;121:593–9. [15] Nowak G, Bucha E. Quantitative determination of hirudin in blood and body fluids. Semin Thromb Hemost 1996;22:197–202. [16] Lange U, Nowak G, Bucha E. Ecarin chromogenic assay–a new method for quantitative determination of direct thrombin inhibitors like hirudin. Pathophysiol Haemost Thromb 2003;33:184–91.
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Contents lists available at ScienceDirect
Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Comparison of monitoring methods for lepirudin: Impact of warfarin and lupus anticoagulant Birgitta Salmela a, Lotta Joutsi-Korhonen b, Ellen Saarela b, Riitta Lassila a,b,⁎ a b
Coagulation Disorders, Division of Hematology, Department of Internal Medicine, Finland HUSLAB Laboratory Services, Helsinki University Central Hospital, Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 23 November 2009 Received in revised form 1 February 2010 Accepted 2 February 2010 Available online 24 February 2010 Keywords: Direct thrombin inhibitor Ecarin Hirudin Lupus anticoagulant Oral anticoagulant
a b s t r a c t Introduction: Appropriate monitoring methods are needed for lepirudin, a direct thrombin inhibitor, as activated partial thromboplastin time (APTT) may under- or overestimate lepirudin. We compared APTT with thrombin-specific methods, also in the presence of warfarin and lupus anticoagulant (LA). Materials and Methods: Lepirudin i.v. was assessed in five patients (35 samples) and in vitro spiked plasma pools: normal control and plasma containing warfarin and LA. Wide dose-responses to lepirudin (0–4.0 µg/ml) were studied with APTT (Actin FSL®), Ecarin Chromogenic Assay (ECA®), chromogenic Anti-Factor IIa (Anti-FIIa, Hirudin Activity Assay®), Prothrombinase-induced Clotting Time (PiCT®), and plasma diluted Thrombin Time (dTT). Results: APTT both under- and overestimated in vivo lepirudin doses according to ECA® and Anti-FIIa, which matched completely in various plasma pools at all lepirudin doses (r= 0.99). APTT and PiCT® underestimated high lepirudin concentrations in normal plasma, and in LA-positive plasma they were invalid. In all plasma pools, dTT (1:16) indicated lepirudin well up to 1.0 μg/ml. Conclusions: ECA® or Anti-FIIa are preferable for lepirudin monitoring, because neither warfarin nor LA, interfered with them, and they were the most precise methods even for supratherapeutic doses. PiCT® reflected co-inhibition of FIIa and FXa, but was disturbed, like APTT, by LA and high lepirudin. Further experience of laboratory monitoring is valuable in this era of new anticoagulants. © 2010 Elsevier Ltd. All rights reserved.
Introduction The recombinant hirudin lepirudin is a potent direct thrombin inhibitor (DTI) [1] which blocks both the active and the fibrinogen binding sites of free and clot-bound thrombin [2,3]. Lepirudin is indicated in heparin-induced thrombocytopenia (HIT) [4] but is also effective in prophylaxis and treatment of thrombosis [5–10]. An appropriate laboratory method for lepirudin monitoring is crucial due to its narrow therapeutic range and high potency. Activated Partial Thromboplastin Time (APTT) is used for monitoring heparin and lepirudin [11], but APTT has poor reproducibility, a non-linear doseresponse, with a plateau even at therapeutic levels, and an inability to detect either lepirudin overdoses or the high concentrations necessary in cardiopulmonary bypass surgery (CPB) [7,12–14].
Abbreviations: APTT, activated partial thromboplastin time; LA, lupus anticoagulant; ECA®, ecarin chromogenic assay; Anti-FIIa, chromogenic Anti-Factor IIa assay; PiCT®, prothrombinase-induced clotting time; dTT, plasma diluted thrombin time; DTI, direct thrombin inhibitor; HIT, heparin-induced thrombocytopenia; CPB, cardiopulmonary bypass surgery; INR, international normalized ratio; AT, antithrombin. ⁎ Corresponding author. Coagulation Disorders, Division of Hematology, Department of Internal Medicine, Helsinki University Central Hospital, P.O. Box 340, 00029 HUS, Helsinki, Finland. Tel.: + 358 40 517 5547; fax: +358 9 471 74504. E-mail address: riitta.lassila@hus.fi (R. Lassila). 0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2010.02.002
Specific direct prothrombin activation methods have been developed for DTI monitoring: ecarin-based methods, e.g. Ecarin Clotting Time (ECT) [15] and Ecarin Chromogenic Assay (ECA®) [16] and Prothrombinase-induced Clotting Time (PiCT®) [17]. Another quantitative measurement, additional to ECA®, is a thrombin-based chromogenic, Anti-Factor IIa (Anti-FIIa) assay [18]. Plasma diluted Thrombin Time (dTT) has been modified from Thrombin Time for lepirudin monitoring [19], though parallel studies of these methods are limited [11,20]. According to our understanding, APTT, ECA®, PiCT®, Anti-FIIa, and dTT have not been compared. The challenges that clinicians face in using APTT are highlighted by our analysis of patient plasma samples obtained during lepirudin treatment. We compared the different methods (APTT, ECA®, PiCT®, Anti-FIIa, and dTT) to assess the intensity of anticoagulation in patient samples and their performance in vitro in assessing lepirudin doseresponses, especially in the presence of warfarin and lupus anticoagulant (LA). Materials and Methods Patient samples Lepirudin has been used at our University Hospital in approximately 50 patients with HIT-related or heparin-resistant thrombosis
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[8–10]. We analyzed 35 plasma samples from five random patients on i.v. lepirudin (Refludan®; Pharmion, Cambridge, UK) (Table 1). Their thrombosis had been treated with unfractionated or low-molecularweight heparin before the switch to lepirudin. Doses of lepirudin had been adjusted on the basis of APTT [21] and of dTT, when available. APTT target ratios were 1.5 to 2.5 depending on the clinical situation. All patients had normal creatinine levels. The study was approved by the hospital's Institutional Review Board and Ethics Committee. Blood samples were collected by antecubital venipuncture into vacuum tubes containing 3.2% sodium citrate (109 mM). Thereafter, plasma was prepared by centrifugation (2,000 ×g, 10 min). Only the upper or middle third of the plasma was collected. After routine laboratory measurements the plasma was stored at -70 °C for less than 12 months. Lepirudin spiking in vitro Plasma was spiked to achieve a wide concentration range of lepirudin (0–4.0 μg/ml). Three different types of pooled plasma were used: 1) Standard Human Plasma (Siemens Healthcare Diagnostics, Marburg, Germany) as normal control plasma containing the standard range of coagulation factors. 2) Commercial warfarin plasmas (Coumadin® plasma, CliniSys Associates, Ltd., USA) at three INR (International Normalized Ratio) levels: 1.5, 2.5, and 3.9 (corresponding to INRs of 1.5, 2.5, and 3.6 provided by CliniSys). 3) Three LA-positive plasma pools (LA1–LA3) were combined from 8 to 21 samples screened positive or strongly positive in two LA tests: the dilute Russell's Viper Venom Time (dRVVT) (DVVtest®, American Diagnostica Inc., Germany) and APTT test (IL Test APTTSP, Instrumentation Laboratory, Italy). Levels of cardiolipin and β2-glycoprotein I antibodies (Varelisa, Cardiolipin IgG Antibodies and Beta-2-Glycoprotein I IgG Antibodies, Phadia GmbH, Freiburg, Germany) were assessed (normal for both b 15 U/ml). In the LApositive plasma pools, antibody levels were 70 and N 100 U/ml (LA1), 40 and 30 U/ml (LA2), and 30 and 50 U/ml (LA3), respectively. The activities of FII (reference range 68–144%), FX (79–146%) (Coagulation Factor II and X Deficient Plasmas, Siemens), and antithrombin (AT; 84–108%) (Berichrom AT III, Siemens) were analyzed before and after addition of lepirudin (1.5 µg/ml) to the plasma pools. The samples were vortex-mixed and immediately studied. Table 1 Lepirudin indications and dosing in patients with proceeding heparin-resistant thrombosis. Patient Age Indication (yrs) 1#
24
2#
28
3#
79
4#
51
5#
38
Pulmonary embolism Hepatic venous thrombosis Critical leg ischemia (inoperable) Critical leg ischemia (inoperable)
Other diagnosis
Dilative cardiomyopathy Paroxysmal nocturnal hemoglobinuria DM, antiphospholipid antibodies DM, CAD, atrial fibrillation, renal transplant, FVIII:c† Critical leg ischemia Horton's neuralgia, (inoperable) with FVIII:c† aortic thrombosis
Median dosage (mg/kg/h) (range)* 0.2 (0.13-0.23) 0.07 (0.05-0.11) 0.06 (0.06-0.09) 0.04 (0.01-0.12)
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Monitoring methods APTT was measured by means of Actin® FSL reagent (Siemens) (reference range 23–33 s and analytic range 8–180 s). For patients #1 to #4, lepirudin therapies had been initially monitored with another APTT reagent (IL Test APTT-SP, reference range 24–40 s). Prothrombin Time (PT) (reference range 70–130%) and INR were studied by the Nycotest® PT reagent (Axis-Shield PoC AS, Oslo, Norway). As specific assays, we used four (see below) monitoring methods for lepirudin. All analyses were performed by a BCS® XP coagulation analyzer (Siemens). Calibrations and controls were performed according to manufacturer's instructions. Ecarin Chromogenic Assay (ECA®) Ecarin, a snake venom metalloprotease, degrades prothrombin and activation products mainly to meizothrombin. An ecarin-based chromogenic assay has earlier been developed for quantifying hirudin (at 405 nm) (HaemoSys® ECA®-H, JenAffin GmbH, Jena, Germany) [16,22]. ECA® prothrombin buffer (100 µl), substrate (25 µl) and the sample (25 µl) were gently mixed and incubated (37 °C, 1 min) and measured after adding ecarin reagent (50 µl). The analytical range was 0–2.0 µg/ml. ECA® is independent of prothrombin and fibrinogen. Both intra- and interassay variations (n = 20, CV%) at 1.5 µg/ml of lepirudin were 7%. Chromogenic Anti-FIIa Assay (Anti-FIIa) An Anti-FIIa assay (Siemens) was used to quantify lepirudin activity in plasma. This method is independent of antithrombin and fibrinogen levels [18]. Plasma sample (25 µl) and substrate (50 µl) were mixed, and the reaction was started with thrombin reagent (250 µl), which induces complex formation 1:1 with lepirudin. Residual thrombin activity was determined by kinetic testing of the increase in absorbance (1 min, at 405 nm). The analytical range was 0–2.3 µg/ml. Intra- and interassay (n = 20) variations were 3–4%. Prothrombinase-induced Clotting Time (PiCT®) The plasma-based functional PiCT® assay (Pefakit®, Pentapharm, Basel, Switzerland) measures anticoagulant activity by FXa or FIIa inhibition, or both [17]. In PiCT®, prothrombinase complex activates coagulation, and the activator reagent contains phospholipids, calcium, FX, and preactivated FV (activator: RVV-V from Russell's viper venom). A two-step coagulation test was performed: incubation (180 s, 37 °C) of plasma (50 µl) with the activator reagent (50 µl) and addition of the start reagent (50 µl 25 mM CaCl2). Clotting time was referred to the standard curve using Pefakit® PICT® Calibrator Hirudin plasmas. The reference range was 19–31 s, and the measurement range was 20–250 s (corresponding to 0–3.0 µg/ml of lepirudin) according to manufacturer's instructions. Intra- and interassay (n = 20) variations were 12%. Plasma Diluted Thrombin Time (dTT) The plasma dTT procedure was developed when TT exceeded 140 s (analytic range 12–140 s) and was too sensitive for therapeutic lepirudin concentrations. TT was performed according to manufacturer's instructions. Buffer solution (5 ml) was added to BC Thrombin reagent® (Siemens) (22 °C, 30 min), plasma (40 µl) was supplemented with BC Thrombin reagent® (100 µl), and clotting time registered up to 150 s. If no clotting was detected, the samples were further diluted with pooled normal plasma. We used serial dilutions from 1:2 to 1:16 until dTT was measurable, or until dilution 1:16.
0.11 (0.1-0.15)
* Lepirudin was started at 0.1–0.15 mg/kg/h without a bolus dose owing to prior anticoagulation therapy. † FVIII:c, factor VIII coagulant activity was high N 200% (normal range 52–148%). DM, diabetes mellitus; CAD, coronary artery disease.
Statistical analysis Statistical analysis was performed with SPSS® 17.01 software (SPSS Inc., Chicago, IL). Intraclass correlation coefficients (ICC; OneWay Random model, with 95% confidence intervals) were determined
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for ECA®, Anti-FIIa, and respective lepirudin concentrations by reliability analysis, whereas Spearman's correlation coefficient (r, 2tailed significance) was used for APTT, PiCT®, and dTT and respective lepirudin concentrations. Significance was set at p b 0.05. Results Monitoring of lepirudin in vivo Patient plasma samples obtained during lepirudin therapy were analyzed with APTT, ECA®, Anti-FIIa, PiCT®, and dTT for dose-responses and possible monitoring difficulties. Both elevated bleeding risk (patients #2 with anemia and thrombocytopenia and #4 with transient renal impairment) and the timing of possible surgery affected treatment strategies in these cases of heparin-resistant thrombosis (Table 1). No clinically significant bleeding occurred. Specific monitoring methods revealed important pitfalls of APTT and the clinical challenges in administering efficacious but safe lepirudin therapy. In an example of overdosing (Fig. 1, patient #1), APTT underestimated lepirudin dosage, whereas ECA®, PiCT®, and Anti-FIIa indicated evident overdosing. The patient was overweight (body mass index 33 kg m-2), and the recommended maximum dosage was nearly exceeded (Table 1). In a patient with paroxysmal nocturnal hemoglobinuria (PNH) and hepatic venous thrombosis (Fig. 1, patient #2), lepirudin dose was kept prophylactic rather than therapeutic, because of anemia and low platelet count. An LA-positive patient's (#3) lepirudin dosing was based on the dTT (1:8 dilution), as
the APTT method was oversensitive (Fig. 1). To avoid overdosing, the infusion was repetitively withheld unnecessarily according to APTT (Actin® FSL), while ECA® and Anti-FIIa indicated underdosing of lepirudin. APTT's insensitivity to dose escalation was seen when it was prolonged only by 5 s versus doubling in ECA® and Anti-FIIa tests (Fig. 1, patient #4). All methods indicated successful dosing in patient #5 (Fig. 1). PiCT®, like APTT, overestimated lepirudin doses in the patients with LA (#3), PNH (#2) and a renal transplant (#4), modestly accumulating lepirudin (Fig. 1). In addition to APTT, dTT (1:8 dilution) was used to guide dosing in patients #1 to #4, but this dilution seemed inadequate for therapeutic lepirudin. At 1:16 dilution, dTT supplemented APTT relatively well (data not shown). In all, inadequate lepirudin dosing, especially in the presence of LA, are evident in APTT responses in comparison with ECA® and Anti-FIIa. Monitoring of lepirudin in vitro Lepirudin concentrations in vitro were chosen to cover the prophylactic, therapeutic (ca. 0.6–1.0 μg/ml), and supratherapeutic levels (2.5–4.0 μg/ml) desired during CPB surgery. Moreover, the combined effects of lepirudin, warfarin-, and LA-positivity were assessed in respective plasma pools. INR and PT (%) The additive anticoagulant effect of lepirudin on INR and PT (%) was measured in three warfarin-containing plasma pools at various
Fig. 1. Monitoring lepirudin treatment in five patients by means of APTT, ECA®, PiCT®, and Anti-FIIa methods. Repetitive plasma samples (on x-axis sample numbers) were obtained during i.v. lepirudin therapy (3-8 days). APTT (s) and respective ECA®, PiCT®, and Anti-FIIa (μg/ml) results in separate panels. Therapeutic range for lepirudin indicated by horizontal lines. Examples of: Overdosing (patient #1): At APTT target, lepirudin concentrations were supratherapeutic according to ECA®, PiCT®, and Anti-FIIa. Underdosing (patient #2): Lepirudin was subtherapeutic according to all methods. Interference by lupus anticoagulant (LA) (patient #3): LA disturbed both APTT and PiCT®, while according to ECA® and AntiFIIa, lepirudin therapy was subtherapeutic. The poor sensitivity of APTT to dose escalation (patient #4): APTT seemed stable despite doubled lepirudin action according to ECA® and Anti-FIIa. Successful laboratory monitoring (patient #5): APTT remained at its target, and ECA® as well as Anti-FIIa indicated appropriate concentrations.
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INR levels (Fig. 2A). At the lowest, INR 1.5, the combined impact was modest even at the highest lepirudin concentration (4.0 μg/ml) (Fig. 2A), but at therapeutic INR levels, lepirudin markedly raised INR. In LA-positive plasma, PT (%) declined with increasing lepirudin concentrations (Fig. 2B), whereas LA had no effect on INRs. APTT Dose-response curves for APTT remained linear only at the lower therapeutic range of lepirudin. Thereafter, APTT reached a plateau in the normal, warfarin-, or LA-positive plasma pool (Fig. 3A, B). APTT was prolonged in all warfarin pools (Fig. 3A). The presence of LA immediately prolonged APTT, resulting in non-measurable clotting, even at low lepirudin concentrations (0.4–0.7 μg/ml) (Fig. 3B). APTT correlated modestly with lepirudin concentrations in different plasma pools (r = 0.69, p b 0.001). ECA® and Anti-FIIa ECA® responses linearly paralleled doses up to 3.0 µg/ml and agreed with spiked lepirudin throughout the dose range (Fig. 4A). Importantly, the presence of warfarin or LA did not disturb ECA® (ICC = 0.99, 95% CI 0.98–0.99, p b 0.001) (Fig. 4A). Anti-FIIa also linearly paralleled lepirudin responses in the various plasma pools. Again, no interference of warfarin or LA was observable (ICC = 0.99, 95% CI 0.985–0.995, p b 0.001) (Fig. 4B). Even the lowest lepirudin
Fig. 2. Responses of INR (A) and PT (B) to lepirudin in vitro in normal, warfarin-, and LApositive plasma pools. Three INR pools (1.5, 2.5, and 3.9) (A) and three LA pools (LA1– LA3) were used (B). Results in normal plasma serve as the reference. Lepirudin and warfarin had an additive effect to INR (A). PT decreased progressively with increasing lepirudin concentration in the presence of LA (B). *Normal refers to normal plasma (at baseline INR was 1.0, PT 96% and LA negative).
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concentrations (0.1 µg/ml) were adequately detected by ECA® and Anti-FIIa methods. These chromogenic methods produced in all plasma pools practically identical results (ICC = 0.99, 95% CI 0.989– 0.996, p b 0.001).
PiCT® PiCT® displayed a relatively linear dose-response to lepirudin in normal plasma in the therapeutic range (r = 0.83, p b 0.001), however, supratherapeutic levels were severely underestimated (Fig. 5A). Coexisting warfarin modified the PiCT® results, even at INR 1.5, precluding separate analysis of lepirudin's anticoagulant effect (Fig. 5A). The presence of LA prolonged PiCT®, like APTT, and PiCT® failed to detect lepirudin in all LA-positive plasma pools (Fig. 5B). Both warfarin and LA interfered with PiCT® (Fig. 5A, B).
dTT As dTT (1:8 dilution) remained below 140 s (i.e. maximum) only at low lepirudin concentrations (b0.7 µg/ml), a further dilution to 1:16 was necessary to extend the measurement range. Responses of dTT to lepirudin up to 1.0 µg/ml appeared relatively linear in normal, warfarin-, and LA-positive plasma pools (Fig. 6A, B). The sensitivity of dTT to increasing lepirudin doses was weaker than with ECA® and Anti-FIIa, but it improved when dTT sample was further diluted from 1:8 to 1:16 (r = 0.93 at 1:8, r = 0.97 at 1:16; both p b 0.001).
Fig. 3. Dose responses of lepirudin according to APTT in vitro in normal (A, B), warfarin(A), and LA-positive (B) plasma pools. Three INR pools (1.5, 2.5, and 3.9) and three LA pools (LA1–LA3) were used. Results in normal plasma served as reference values. APTT reached a plateau in normal plasma (A, B). Warfarin at INR 2.5 and 3.9 together with lepirudin prolonged APTT (A). LA extensively prolonged APTT (B).
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Fig. 4. Dose responses of lepirudin according to ECA® (A) and Anti-FIIa (B) in vitro in normal plasma, warfarin-, and LA-positive plasma pools. Three INR pools (1.5, 2.5, and 3.9) and three LA pools (LA1–LA3) were used. Neither warfarin nor LA interfered with the linear dose-responses of ECA® or Anti-FIIa.
Comparison of monitoring methods Normal control plasma APTT showed no linear dose-response at lepirudin concentrations above 0.5 μg/ml (Fig. 3A). In contrast, both ECA® and Anti-FIIa data were linear throughout the wide concentration range (Fig. 4). Importantly, ECA® and Anti-FIIa methods detected supratherapeutic lepirudin concentrations agreeing with the spiking, in contrast to PiCT®, which indicated 20% lower concentrations of lepirudin in the upper therapeutic range (Fig. 7). In addition, dTT (1:16 dilution) well assessed the dose-responses of lepirudin up to 1.0 μg/ml (Fig. 6A). FII, FX, and AT activities were normal with and without lepirudin (1.5 μg/ ml). Warfarin-containing plasma The co-effect of lepirudin and warfarin prolonged APTT without any significant difference at an INR between 2.5 and 3.9 (Fig. 3A). Both ECA® and Anti-FIIa methods detected lepirudin doses linearly regardless of distinct vitamin K-dependent coagulation factor deficiencies (Fig. 4). PiCT®, however, indicated higher levels, and was progressively distorted by warfarin at INR ≥1.5 and lepirudin above 0.7 μg/ml (Fig. 5A). An increasing warfarin concentration (INR) seemed not to affect dTT (1:16 dilution) results (Fig. 6A). As expected, FII activities were low, paralleling INR at baseline, and were further diminished after addition of lepirudin (1.5 μg/ml) (INR 1.5: from 53 to 37%, INR 2.5: from 28 to 10%, and INR 3.9: from 19 to 8%). After addition of lepirudin (1.5 μg/ml) the activities of FX remained at the same low levels (INR 1.5: from 41 to 36%, INR 2.5: 11% and INR 3.9: 9%), but AT remained normal (data not shown).
Fig. 5. Dose responses of lepirudin according to PiCT® in vitro in normal plasma (A, B), warfarin-, (A) and LA-positive (B) plasma pools. Three INR pools (1.5, 2.5, and 3.9) and three LA pools (LA1–LA3) were used. Results in normal plasma served as reference values. Coexisting warfarin clearly prolonged PiCT®, even at INR 1.5 (A). The presence of LA uniformly prolonged PiCT® (B).
LA-positive plasma Both APTT and PiCT® showed prolonged coagulation times in the presence of LA, and lepirudin could not be traced (Figs. 3B, 5B). In contrast, ECA® and Anti-FIIa predicted lepirudin doses in LA plasma (Fig. 4). The three LA-positive pools produced similar dTT data (Fig. 6B). Lepirudin (1.5 μg/ml) somewhat reduced FII activity (LA1: 65%, LA2: 63%) in LA-positive pools, whereas FX and AT activities remained normal. Discussion Currently, as a result of the lack of a reliable monitoring method, the antithrombotic potential of lepirudin may be underused regarding therapy and CPB surgery. According to our results both chromogenic methods were precise and appeared suitable for monitoring of lepirudin-induced anticoagulation, even including supratherapeutic levels. Importantly, ECA® and Anti-FIIa were reproducible and unaffected by warfarin or LA, common clinical confounding factors. In contrast, APTT underestimated lepirudin in the upper therapeutic range and appeared unsuitable in the presence of warfarin or LA. Unfortunately, PiCT® seemed to show some of the same limitations. Although ECA® and Anti-FIIa methods provide accurate options for lepirudin monitoring, a functional method could indicate residual inhibition of coagulation when bridging with other anticoagulants (i.e. heparin and warfarin). Additionally, lepirudin may produce distinct responses in coagulation assays during thrombosis and fibrinolysis. APTT is severely limited, as it may indicate falsely high lepirudin levels
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Fig. 6. Dose responses of lepirudin in vitro according to dTT (1:16 dilution) in normal plasma (A, B), warfarin- (A), and LA-positive (B) plasma pools. Three INR pools (1.5, 2.5, and 3.9) and three LA pools (LA1–LA3) were used. Results in normal plasma served as reference values. The presence of warfarin (A) and LA (B) had no effect on dTT. † Lowest lepirudin concentrations (0 and 0.1 µg/ml) are presented with undiluted TT, whereafter, TT became unmeasurable without further dilution.
under several clinical situations, including prothrombin deficiency, liver disease, disseminated intravascular coagulation, or the presence of LA, or vitamin K antagonists [7]. In the case of warfarin, our findings confirm the previous observations [23,24] in a wider range of INR (1.5-3.9) and analytical methods. For instance, in HIT, a premature disruption of lepirudin infusion due to falsely high APTT may be
Fig. 7. Comparison of dose responses of lepirudin by ECA®, Anti-FIIa, and PiCT® methods in normal plasma. ECA® and Anti-FIIa produced relative similar, exact, and linear dose responses throughout the wide concentration range of lepirudin (0-4.0 µg/ml). PiCT® underestimated lepirudin concentrations above 1.0 µg/ml.
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deleterious during thrombosis and possible “rebound” thrombin generation [7]. The risk that lepirudin tapering would be unnecessary could be avoided by use of assays like ECA® and Anti-FIIa, both independent of prothrombin or fibrinogen, and of the presence of LA and warfarin. PiCT underestimated lepirudin in the upper therapeutic range in normal plasma in comparison with ECA® and Anti-FIIa (Fig. 7), as noted earlier [13], the underlying reason remaining speculative. On the other hand, the enhanced warfarin effect on PiCT® could signal the presence of high lepirudin doses, when bridging oral anticoagulants and vice versa. In case of LA-positivity, however, PiCT®, like APTT, is invalid. Besides APTT, if ECA® or Anti-FIIa methods are unavailable, dTT could help in adjusting appropriate lepirudin dosing, as suggested also by others [19]. In the therapeutic range of lepirudin, dTT at 1:8 exceeds the measurement range (140 s), whereas dTT at 1:16 should remain under a specified limit (in our case ∼ 50 s), to be assessed by each laboratory. The anticoagulant effect of lepirudin is more predictable than that of heparin due to the lack of cofactors, to non-specific binding characteristics onto cellular surfaces, and to neutralization by proteins [25,26]. Lepirudin has a short plasma half-life (1.5–2 h), but on the clot surface and in the extravascular space it remains active far longer [3], making rebound-like action plausible. Coagulation activity is inhibited at low concentrations, whereas higher concentrations are needed to inhibit development of platelet-dependent arterial thrombosis [25,27]. Safe and effective treatment with lepirudin demands a reliable monitoring method to avoid under- and overdosing; especially during warfarin therapy or its initiation after lepirudin or in the presence of LA. Moreover, in association with impaired renal function the half-life of lepirudin may be prolonged, even up to 30fold [28,29]. Small but effective doses of lepirudin can be administered, according to ECA® or Anti-FIIa. In our patients, lepirudin dosing turned out to be less than intended (Table 1), but deliberate underdosing (patients #2 to #4) occurred due to the limitations of APTT. Indeed, recently published data and guidelines recommend lower than earlier lepirudin dosing levels [4,7,21,30–32]. The ultimate target is to avoid hemorrhagic problems, while achieving appropriate therapeutic inhibition of thrombin in pharmacodynamically vulnerable patients. Both ECA® and Anti-FIIa methods can be adapted for automated analyzers. Anti-FIIa reagents are stable and may be stored longer than ECA® reagents. ECA® is also feasible for point-of-care testing. Clinically, bedside methods would be useful, especially during CPB surgery. ECT is more widely used than ECA®, as it has been long available. ECT shows a linear association with lepirudin, even at a higher concentration than ECA® (up to 5.0 µg/ml) [15]. Both ECT and ECA® are independent of LA, warfarin, and fibrinogen, but ECA® overcomes the limitation of ECT during prothrombin deficiency [16,22]. The importance of comparative monitoring data on various DTIs and FXa inhibitors is increasing as many new anticoagulants, i.e. dabigatran and rivaroxaban, are emerging [33,34]. During overdosing, severe bleeding complication, emergency surgery, or under renal insufficiency, a drug-specific and reliable monitoring method augments appropriate management. Our study results apply to lepirudin only, and for other DTIs the dose-responses need to be individually studied. ECA®, however, seems to provide a linear dose-response also with other DTIs, i.e. argatroban and bivalirudin (according to manufacturer's instruction), in contrast to APTT, PiCT®, and ECT [13,22,35,36]. In conclusion, automated quantitative analysis of lepirudin by means of ECA® or Anti-FIIa assays can be recommended to improve safety in patients treated with the potent DTI lepirudin, even during CBP surgery. When lepirudin is administered with oral anticoagulants or heparins, PiCT® or APTT together with chromogenic ECA® or AntiFIIa methods may be helpful in assessing prevailing anticoagulant
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