Thrombosis Research 134 (2014) 918–923

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Regular Article

Measuring the activity of apixaban and rivaroxaban withrotational thrombelastometry☆

Dieter Adelmann a, Marion Wiegele a, Rudolf Karl Wohlgemuth a, Stefan Koch a, Sophie Frantal b,Peter Quehenberger c, Gisela Scharbert a, Sibylle Kozek–Langenecker d, Eva Schaden a,⁎a Department of Anaesthesia, General Intensive Care and Pain Control, Medical University of Vienna, Vienna, Austriab Section for Medical Statistics, Medical University of Vienna, Vienna, Austriac Department of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austriad Department of Anaesthetics and Intensive Care, Evangelical Hospital Vienna, Vienna, Austria

Abbreviations: FXa, factor Xa; PT, prothrombin time; Ptational thrombelastometry®; LowTF, low–tissue factor; Pclotting time®; DMSO, dimethylsulfoxide; CT, clotting tiMaxVel, time tomaximumvelocity; A10, clot amplitude atation; CI, lower and upper 95% confidence interval; SCC, spROCs, receiver operating characteristic curves.☆ Abstracts of this work have been presented in prelimiing of the Society of Thrombosis and Haemostasis Researcthe annual meeting of the European Society of Anaesthes⁎ Corresponding author at: Medical University of Vien

General Intensive Care and Pain Control, WaehringerAustria. Tel.: +43 1 40400 4150; fax: +43 1 40400 4028

E-mail address: eva.schaden@meduniwien.ac.at (E. Sc

http://dx.doi.org/10.1016/j.thromres.2014.08.0060049-3848/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 16 September 2013Received in revised form 28 July 2014Accepted 10 August 2014Available online 19 August 2014

Keywords:ApixabanBlood coagulation testsPoint-of-Care coagulation testsRivaroxabanThrombelastography

Background: Routine drug monitoring is not required for the two novel direct factor Xa inhibitors apixaban andrivaroxaban. Rapidly available test results might be beneficial in case of bleeding or prior to urgent surgery.Objectives: The aim of this study was to evaluate the applicability of the two rotational thrombelastometry(ROTEM®) -modifications Low–tissue factor activated ROTEM® (LowTF–ROTEM®) and Prothrombinase in-duced clotting time – activated ROTEM® (PiCT®–ROTEM®) for determination of apixaban and rivaroxabanin vitro and ex vivo.Methods: Blood samples from 20 volunteers were spiked with apixaban / rivaroxaban to yield samples withascending drug concentrations ranging from 50 – 400 ng/mL. LowTF – and PiCT® modified ROTEM® tests anddetermination of the corresponding antifactor Xa activity were performed in duplicate in 280 samples. LowTF–ROTEM® tests were performed in samples from 20 patients on apixaban or rivaroxaban therapy and 20 controls.Results: There was a strong correlation between apixaban / rivaroxaban plasma concentrations and the LowTF–

ROTEM® parameters Clotting time (CT; spearman correlation coefficient (SCC) 0.81 and 0.81, respectively)and Time to maximum velocity (t,MaxVel; SCC: 0.81 and 0.80, resp.) and a low to moderate correlation for thePiCT®–ROTEM® parameters CT (SCC: 0.38 and 0.59, resp.) and t,MaxVel. (0.51 and 0.69, resp.) in the in vitro ex-periments. LowTF–ROTEM CT was significantly prolonged in patients on apxiaban or rivaroxaban therapy com-pared to controls.Conclusions: LowTF–ROTEM®could be a valuable diagnostic tool for rapid determination of the effect of apixabanand rivaroxaban at the point of care.

© 2014 Elsevier Ltd. All rights reserved.

Introduction

The direct factor Xa (FXa) inhibitors apixaban and rivaroxabanpermit the use of fixed dosing schemes without the need for doseadjustments based on coagulation tests. However, rapidly availabletests might be beneficial in situations such as thrombotic events or

OC, point of care; ROTEM®, ro-iCT®, prothrombinase inducedme; CFT, clot formation time; t,10 minutes; SD, standard devi-earman correlation coefficient;

nary format at the annual meet-h (Munich, February 2013) andiology (Barcelona, June 2013).na, Department of Anaesthesia,Guertel 18–20, 1090 Vienna,.haden).

bleeding complications, the need for urgent surgery or suspected drugoverdose [1,2]. Routine coagulation tests are not recommended formonitoring of direct FXa inhibitors. Although prothrombin time (PT)is influenced by rivaroxaban, its sensitivity depends on the thrombo-plastin reagent used [3]. Chromogenic anti-FXa tests allow quantifica-tion of rivaroxaban plasma levels but they are not suitable for point ofcare (POC) testing [4]. No suitable method for POC drug monitoringfor apixaban or rivaroxaban has yet been described [1].

Rotational thrombelastometry (ROTEM®) is a viscoelastic coagula-tion test that allows rapid detection of coagulation abnormalitiesat the point of care [5]. Conventional ROTEM® tests are insensitive to di-rect FXa inhibitors such as rivaroxaban [6].

Two new ROTEM® test modifications have previously been shownto be sensitive for indirect FXa inhibition by unfractionated heparinand enoxaparin [7,8]. Low–tissue factor activated ROTEM® (LowTF–ROTEM®) utilizes minimal amounts of tissue factor as a coagulationtrigger and has an increased sensitivity for coagulation factor deficienciesand anticoagulant therapy [9]. Prothrombinase induced clotting time –

919D. Adelmann et al. / Thrombosis Research 134 (2014) 918–923

activated ROTEM® (PiCT®–ROTEM®) is based on the PiCT® assay, aclotting assay sensitive to FXa and Factor IIa inhibition [10].

Wehypothesized that thesemodified ROTEM®assays also detect di-rect FXa inhibition by rivaroxaban and apixaban. The objective of thisstudy was to evaluate the applicability of LowTF– and PiCT®–ROTEM®for POC determination of apixaban and rivaroxaban.

Materials and Methods

The studywas approved by the ethics committee of theMedical Uni-versity of Vienna. (EK 571/2010) Blood samples from three differentpopulations were analyzed: For the apixaban and rivaroxaban in vitroexperiments, blood samples from 20 volunteers were included (“volun-teer group”). For the ex vivo measurements, samples from patients onapixaban (minimum dose: 2.5 mg twice daily) or rivaroxaban (mini-mum dose: 15 mg once daily) therapy were included in the “patienttherapy group”. In addition, 20 patients presenting to the Departmentof Trauma Surgery with fractures of the femur neck or undergoing cere-bral computer tomography after minor head trauma who were not onanticoagulant medication were included as “patient control group”.

Exclusion criteria were abnormal bleeding history [11], antiplateletor anticoagulant medication intake 14 days prior to admission (volun-teer and patient control groups only), abnormal platelet count or rou-tine coagulation parameters.

Venous bloodwas drawn from the cubital veinwithout stasis using a21–gauge butterfly needle. Forty–three millilitres (mL) of blood weredrawn from each volunteer: the first 3 mL were discarded, 3 mL weredrawn into a potassium EDTA tube (Vacuette™, Greiner, Kremsmünster,Austria) for blood cell counts and 1 mL was drawn into a heparinizedsyringe (Radiometer Medical, Brønshøj, Denmark) for haematocritmeasurement. Thirty–six mL of blood were drawn into four 9 mL tubescontaining trisodium citrate 3.8% (Vacuette™ Greiner, Kremsmünster,Austria; 9:1 v/v) for routine coagulation testing and rivaroxaban orapixaban sample preparation.

Immediately after withdrawal, the EDTA tube and 2 mL of citratedwhole blood were sent to the hospital’s central laboratory. Completeblood counts were assessed with a Sysmex XE– 2100 cell counter(Sysmex, Kobe, Japan). After centrifugation PT, aPTT and the fibrinogenlevelwere determinedusing an automated coagulation analyzer. (STA –REvolution® coagulometer; Diagnostica Stago S.A.S., Asnières sur Seine,France).

Sample Preparation

Apixaban was provided by Bristol–Myers Squibb (Eliquis®, NewYork, United States). Rivaroxaban was provided by Bayer (Xarelto®,Berlin, Germany). Four milligrams (mg) of apixaban and 4 mg ofrivaroxaban were each dissolved in 10 mL of 100% dimethylsulfoxide(DMSO) to yield stock solutions with an apixaban or rivaroxaban con-centration of 400 micrograms (μg)/mL. Five working solutions with de-creasing apixaban / rivaroxaban concentrations were prepared throughdilution of the stock solutions in albumin 5%.Whole blood samplesweredivided into aliquots of 4 mL and spiked with apixaban or rivaroxabanworking solutions.

According to previous studies on clinically relevant plasma concen-trations [12,13], targeted concentrations (C) in our in vitro experimentswere 50 ng/mL (C–1), 100 ng/mL (C–2), 200 ng/mL (C–3), 300 ng/mL(C–4) and 400 ng/mL (C–5) for apixaban and rivaroxaban. Due to thehigh plasma protein binding of apixaban and rivaroxaban (87% and92% – 95%, respectively) [14,15] the use ofwhole blood required the ad-justment of the amount of working solution to the calculated plasmavolume of each volunteer. The mean volume of the working solutionadded per sample was 33 μL. The maximal DMSO concentration inspiked whole blood samples was 0.08% for the apixaban experimentsand 0.09% for the rivaroxaban experiments.

Two samples were used as controls: one sample of 2 mL citratedwhole blood was used for baseline ROTEM® testing (control sample1) and one sample was spiked with the working solution (DMSO in 5%Albumin; 1:9) alone (control sample 2) to control for possible effectsof the working solution on ROTEM® test results.

All 5 spiked samples and the 2 control samples were incubated for20 minutes at 36 °C while gently moved. Two mL of each samplewere used for ROTEM® analysis. The residue was centrifuged at1450 G and 4 °C for 10 minutes to obtain plasma which was stored at−80 °C for subsequent chromogenic FXa testing.

In the two patient groups, LowTF–ROTEM®and chromogenic antiXaconcentration measurements were determined from blood samplesdrawn in 4 mL tubes containing trisodium citrate 3.8% (Vacuette™Greiner, Kremsmünster, Austria; 9:1 v/v) during routine blood analysis.

FXa Chromogenic Tests

For quantitative measurement of apixaban and rivaroxaban concen-trations, the chromogenic test BIOPHEN DiXaI (Hyphen–Biomed,Neuville–sur–Oise, France) was used [16]. Testing was performed onthe STA –R Evolution® coagulometer (Diagnostica Stago S.A.S., Asnièressur Seine, France). For apixaban, calibrators (at 250 ng/mL and500 ng/mL) and control samples (at 100 ng/mL and 300 ng/mL) werecreated in duplicate by spiking control plasma (Hyphen–Biomed,Neuville–sur–Oise, France) with two separatelymanufactured stock so-lutions of apixaban. For rivaroxaban, calibrators and control sampleswere obtained from Hyphen–Biomed.

Rotational Thrombelastometry

Rotational Thrombelastometry was performed on a ROTEM® ana-lyzer (TEM Innovations, Munich, Germany) within two hours afterblood samples were drawn.

Low tissue factor –modified ROTEM® (LowTF–ROTEM®) [9] utilizesminimal amounts of tissue factor as an activator. Lyophilized recombi-nant tissue factor (Dade Innovin®, Siemens Healthcare, Marburg,Germany)was reconstitutedwith distilledwater using the volume stat-ed on the vial label (4 mL). One hundred μL of this stock solution werediluted with 100 mL of distilled water to a ratio of 1 : 1000. Twenty μLof the resulting solution were incubated with 300 μL of citrated wholeblood and coagulation was started through recalcification with 20 μLof calcium chloride (CaCl2) 0.2 mol/L. Thus, the dilution of tissue factorin the final sample volume of 340 μL was 1 : 17 000 (~0.350 pmol/L).Stability of the stock solution is guaranteed for 10 days; further dilu-tions were prepared accordingly.

Prothrombinase induced clotting time –modified ROTEM® (PiCT®–

ROTEM®) utilizes PiCT® reagent, an assay sensitive to FXa – and factorIIa – inhibitors [10]. The PiCT® reagent (Pefakit PiCT®; Pentapharm,Basel, Switzerland) was reconstituted according to the manufacturer’sinstructions. The resulting solution was aliquoted, stored at −20 °C,and allowed to thaw immediately before analysis. Twenty μL of thePiCT reagent were incubated with 300 μL of citrated whole blood for180 s and coagulation was started subsequently through recalcificationwith 20 μL of CaCl2 0.2 mol/L.

The two ROTEM® test modifications LowTF–ROTEM® and PiCT®–

ROTEM®were performed in duplicate measurements. ROTEM® qualitycontrol measurements were performed once weekly as recommendedby the manufacturer.

The main endpoints were Clotting time (CT) and Time to maxi-mum velocity (t,MaxVel) which give information about the initialactivation of clot formation and are prolonged by anticoagulants[9]. Time to maximum velocity is a ROTEM® parameter not usedin clinical practice. It is defined as the time to maximum velocityof clot formation on the first derivative of the ROTEM® curve. Clot for-mation time (CFT) and clot amplitude at 10 minutes (A10) served assecondary endpoints.

920 D. Adelmann et al. / Thrombosis Research 134 (2014) 918–923

Statistical Analysis

Descriptive results (mean+/− standard deviation (SD) or frequen-cies and percentages) are given for all variables. To describe consistencyof the two repeated measurements per sample the intraclass–correla-tion–coefficient with the according 95% confidence interval (CI) wascalculated. Since consistency was good, all following analyzes weredone using the mean value of the two repeated measures per patient,reagent and concentration. Spearman rank correlation coefficients(SCC) were used to describe the correlation between CT, CFT, t,MaxVeland A10 with antiXa apixaban and rivaroxaban concentrations, respec-tively. Comparisons between concentrations were primarily done in avisual way using boxplots. To compare the results of the two controlgroups in CT, CFT, t,MaxVel and A10 paired t–tests were used. For thecut–off values of antiXa apixaban and rivaroxaban concentrations of0 ng/mL and 200 ng/mL, receiver operating characteristic curves(ROCs) were calculated and presented for CT and t,MaxVel. Areasunder the ROCs with 95% confidence intervals and optimal cut–offs foreach variable are shown. To compare parameters within the patientsgroups, independent sample t-tests were used.

Analysis was performed using R 2.12.2. All p–values b 0.05 are con-sidered as statistically significant. No correction for multiplicity wasdone.

Results

Baseline values of LowTF– and PiCT®–ROTEM® parameters of thevolunteers and the patient control group are shown in Table 1.

Apixaban

Apixaban ConcentrationsFor the 5 spiked whole blood samples, the following mean chromo-

genic antiXa apixaban concentrationswere determined via chromogen-ic testing calibrated for apixaban: C1: 50 ng/mL (±10 ng/mL), C2:120 ng/mL (±10 ng/mL), C–3: 250 ng/mL (±30 ng/mL), C–4:300 ng/mL (±30 ng/mL) and C–5: 420 ng/mL (±20 ng/mL).

Correlation and ConsistencyCorrelations between chromogenic antiXa apixaban concentrations

and ROTEM® parameters and the intraclass correlation coefficient forthe duplicate measurements are shown in Table 2. Boxplots for theLowTF– ROTEM® parameter CT are shown in Fig. 1.

Cut–off Values

Cut–off values for chromogenic antiXa apixaban concentrations of0 ng/mL and 200 ng/mL were calculated for the LowTF–ROTEM® pa-rameters CT and t,MaxVel. Cut–off values are given in Table 3.

Rivaroxaban

Rivaroxaban ConcentrationsThe following mean chromogenic antiXa rivaroxaban concentrations

were determined via chromogenic testing calibrated for rivaroxaban for

Table 1Baseline values of LowTF– and PiCT®–ROTEM® parameters in the volunteer (n = 40)group [mean (±SD)].

LowTF–ROTEM® PiCT®–ROTEM®

Mean (±SD) Mean (±SD)

CT (s) 318 (±69) 147 (±19)t,MaxVel (s) 382 (±89) 189 (±28)CFT (s) 108 (±45) 79 (±22)A10 (mm) 53 (±7) 57 (±5)

the 5 spiked whole blood samples: C–1: 60 ng/mL (±20 ng/mL),C–2: 160 ng/mL (±10 ng/mL), C–3: 200 ng/mL (±20 ng/mL), C–4:290 ng/mL (±30 ng/mL and C–5: 420 ng/mL (±30 ng/mL).

Correlation and ConsistencyCorrelations between chromogenic antiXa rivaroxaban concentra-

tions and ROTEM®parameters and the intraclass correlation coefficientfor the duplicate measurements are shown in Table 2. Boxplots for theLowTF– ROTEM® parameter CT are shown in Fig. 2.

Cut–off Values

Cut–off values for chromogenic antiXa rivaroxaban plasma concen-trations of 0 ng/mL and 200 ng/mL were calculated for the LowTF–ROTEM® parameters CT and t,MaxVel. Cut–off values are given inTable 3.

Patient Samples

In the patient therapy group, the mean time between last time ofdrug intake and blood withdrawal was 3.81 hours (±2.57 hours). Themean chromogenic antiXa concentration was 64 ng/mL (±56 ng/mL)for apixaban and 535 ng/mL (±147 ng/mL) for rivaroxaban. In patientson rivaroxaban therapy, the LowTF–ROTEM® parameters CT, CFT, A10,MaxVel and t,MaxVel were significantly prolonged compared to the pa-tient control group (p b 0.05). In patients on apixaban therapy LowTF–CT was significantly prolonged compared to the patient control group(p b 0.05). ROTEM parameters are shown in Table 4.

Discussion

Drug monitoring of direct FXa inhibitors is generally not required. Apoint of care test for the drug effect of apixaban and rivaroxaban couldprovide useful information in critical situations such as urgent surgery,when the risk of blood loss must be assessed prior to surgery or in pa-tients suffering from stroke while on direct FXa inhibitor therapy [1,2,17].

In the present study, apixaban and rivaroxaban both significantlyprolonged ROTEM® CT and t,MaxVel when diluted tissue factor wasused as a trigger. This increase in CT and t,MaxVel strongly correlatedwith the chromogenic antiXa concentrations of apixaban and riva-roxaban. The in-vitro findings were confirmed in samples from 20 pa-tients on apixaban and rivaroxaban therapy. LowTF–ROTEM® CT wassignificantly longer when compared to patients who were not on anti-coagulant therapy. There was no difference in correlation coefficientsbetween CT and the t,MaxVel, therefore the alreadywell established pa-rameter CT is sufficient. There was only minimal influence on the clotamplitude, as has been previously described for oral anticoagulants[18]. The correlation between the PiCT®–ROTEM® parameters CT andt,MaxVel and apixaban and rivaroxaban concentrations was less pro-nounced than for LowTF–ROTEM® parameters.

Although there was overlap of clotting times between the controlgroups and low concentrations of apixaban and rivaroxaban (Figs. 1and 2) cut-off values for LowTF–CT and apixaban and rivaroxaban con-centrations of 200 ng/mL could be calculatedwith a high sensitivity andspecificity for the in-vitro data. This overlap and the high standard devi-ation of LowTF–CT results precludes the use of LowTF–ROTEM® forquantitative determination of apixaban and rivaroxaban plasma con-centrations. Nevertheless the use as a semi-quantitative test is of inter-est for preoperative screening, especially in in urgent situations.

Point of care testing such as viscoelastic testing is immediately acces-sible in many acute care and perioperative settings. Thrombelastometrycan be performed at the point of care in whole blood without the needfor further preanalytical procedures. Commercially available viscoelastictests such as EXTEM® do not seem to provide adequate sensitivity fordetermination of the new direct FXa inhibitors [6,19]. Compared to high

Table 2Correlation between chromogenic antiXa apixaban and rivaroxaban concentrations and ROTEM® parameters (spiked blood samples, volunteer group).

LowTF–ROTEM® PiCT®–ROTEM®

Spearman correlationcoefficient (p-value)

Intraclass correlationcoefficient (CI)

Spearman correlationcoefficient (p-value)

Intraclass correlationcoefficient (CI)

ApixabanCT (s) 0.81 (b0.01) 0.96 (0.95 – 0.97) 0.38 (b0.01) 0.60 (0.48 – 0.69)t,MaxVel (s) 0.81 (b0.01) 0.95 (0.93 – 0.96) 0.51 (b0.01) 0.60 (0.48 – 0.70)CFT (s) 0.68 (b0.01) 0.92 (0.89 – 0.94) 0.14 (0.10) 0.64 (0.53 – 0.73)A10 (mm) −0.40 (b0.01) 0.95 (0.93 – 0.96) 0.19 (0.03) 0.80 (0.74 – 0.86)

RivaroxabanCT (s) 0.81 (b0.01) 0.95 (0.93 – 0.96) 0.59 (b0.01) 0.84 (0.79 – 0.88)t,MaxVel (s) 0.80 (b0.01) 0.94 (0.92 – 0.96) 0.69 (b0.01) 0.83 (0.77 – 0.87)CFT (s) 0.56 (b0.01) 0.84 (0.79 – 0.89) 0.30 (b0.01) 0.62 (0.51 – 0.72)A10 (mm) −0.34 (b0.01) 0.89 (0.85 – 0.92) 0.04 (0.65) 0.86 (0.81 – 0.90)

921D. Adelmann et al. / Thrombosis Research 134 (2014) 918–923

tissue factor dependent activation in EXTEM®, low tissue factor activatedthrombelastometry can increase the sensitivity of thrombelastometrictests for coagulation factor deficiency and anticoagulant therapy [9]. Asimilar concept of diluted thromboplastin reagents for PT testing hasbeen proposed for the determination of rivaroxaban [18].

A particular advantage of LowTF–ROTEM® tests would be its rapidavailability of test results at the point of care [20]. LowTF–ROTEM® CTresults are available within 15 minutes after the blood sample isdrawn. Chromogenic AntiXa assays might not be readily available out-side regular working hours at many institutions. Sample transportationto a central laboratory and turn-around times might further delay thetimely availability of test results. LowTF–ROTEM® results might not beas precise as chromogenic AntiXa assays; however, the rapid availabilityof this test could be of great clinical value for the acute care physician.

Despite the moderate correlation found for PiCT®–ROTEM® CT andt-max,Vel, a biphasic pattern with an initial shortening of CT times atthe lowest concentrations (50 and 100 ng/mL, data not shown) ofrivaroxaban and apixaban was observed. Similar to our previous inves-tigations, we utilized a two-step assay for PiCT®–ROTEM® testing withan incubation period of 180 s prior to recalcification. A comparable ef-fect of initial clotting time shortening in the determination of directFXa inhibitors was reported for the conventional two-step PiCT®assay [21,22]. This initial shortening, the low correlation coefficientand the low intraclass correlation coefficient of the PiCT®–ROTEM® re-sults preclude the use of the two-step PiCT®–ROTEM®assay for the de-termination of direct FXa inhibitors.Whether a one-step PiCT®assay for

Apixaban concentration (ng/mL)

40030020010050Control_2Control_1

Low

TF

Clo

ttin

g T

ime (

seconds)

1400

1200

1000

800

600

400

200

0

Fig. 1. LowTF–ROTEM® Parameters for Apixaban; CT for LowTF–ROTEM ®; Control_1:control sample 1 (whole blood), Control_2: control sample 2 (whole blood + workingsolution).

thrombelastometry could be utilized for apixaban and rivaroxaban test-ing will have to be evaluated in a further study [23].

The following limitations of this study have to be considered. Extrap-olations from in–vitro experiments to clinical practice must be madewith care. We only included blood samples from healthy volunteers inour spiking experiments. Although this might be seen as a limitationof our study, an in-vitro model using samples from healthy volunteersallows for better standardization and therefore reduces the confoundingeffects of concomitant medication intake and pre-existing diseases inblood samples from patients. This might outweigh some of the limita-tions of the chosen method.

Awide variability in LowTF–ROTEM®parameterswas observed, thiswould limit the utility for quantitative interpretation of the test results.However, LowTF–ROTEM®can yield semi quantitative information on apossible drug effect, which can be of value to the acute care physician.All samples were collected in 3.8% sodium citrate tubes. Although thechoice of 3.2% or 3.8% sodium citrate has previously been shown notto influence antiXa levels of low molecular weight heparin we can notexclude an effect of sodium citrate on ROTEM® parameters or antiXaapixaban and rivaroxaban levels [23].

Major blood loss and subsequent hemodilution might influencethrombelastometric as well as conventional coagulation tests. LowTF–and PiCT®–ROTEM® results must be interpreted in the clinical context,and more specific tests such as chromogenic antiXa tests should beapplied as a confirmatory test for the presence of Factor Xa inhibitorsas soon as clinically available.

Despite the promising results of these proof of concept experimentsLowTF–ROTEM® testing should not be directly applied in clinical prac-tice. Further adequately powered clinical studies are necessary.

We presented an in–vitro spiking model of whole blood samples.Previous in–vitro studies evaluating laboratory test for novel anticoagu-lants have utilized spiking of plasma samples [24,25]. Due to their low

Table 3Cut–off values for apixaban and rivaroxaban (LowTF–ROTEM®, spiked blood samples, vol-unteer group).

Parameter Time Sensitivity Specificity

Apxiaban : 0 ng/mLCT (s) 432 0.96 0.97t,MaxVel (s) 569 0.92 1.00

Apixaban : 200 ng/mLCT(s) 548 0.95 0.74t,MaxVel (s) 621 1.00 0.69

Rivaroxaban : 0 ng/mLCT (s) 426 s 0.90 0.88t,MaxVel (s) 521 s 0.89 0.88

Rivaroxaban : 200 ng/mLCT (s) 524 s 0.98 0.69t,MaxVel (s) 658 s 0.92 0.72

Rivaroxaban concentration (ng/mL)

40030020010050Control_2Control_1

Low

TF

Clo

ttin

g T

ime (

seconds)

1400

1200

1000

800

600

400

200

0

Fig. 2. LowTF–ROTEM® Parameters for Rivaroxaban; CT for LowTF–ROTEM®; Control_1:control sample 1 (whole blood), Control_2: control sample 2 (whole blood + workingsolution).

922 D. Adelmann et al. / Thrombosis Research 134 (2014) 918–923

solubility, apixaban and rivaroxaban must both be dissolved in thesolvent DMSO. The effect of DMSO on coagulation parameters orthrombelastometric parameters after whole blood sample spiking hasnot been evaluated in previous studies. For the apixaban spiking exper-iments, there was no significant difference in thrombelastometric pa-rameters between the whole blood samples and the control samplecontaining DMSO. For the rivaroxaban experiments, an significant in-crease in CT and t,MaxVel (17% and 20%, respectively) was observed.The DMSO content of the control sample was 0.09%, equivalent to theDMSO content of sample 5 (rivaroxaban concentration: 400 ng/mL).Due to the further dilution of the subsequent working solutionsused to spike the samples with lower rivaroxaban concentrations, theDMSO content in these samples is reduced to a minimum of 0.007% insample 1 (50 ng/mL). Despite this low DMSO concentration the influ-ence of the solvent on ROTEM® parameters in the spiking experimentscannot be ruled out.

Conclusion

We could demonstrate a dose dependent effect of apixaban andrivaroxaban on LowTF–ROTEM® test parameters CT and t,MaxVelin vitro. LowTF–ROTEM® CT was significantly prolonged in patientson apixaban and rivaroxaban therapy. LowTF–ROTEM®, in addition tothe detailed bleeding history [11] might permit rapid determination ofa drug effect in patients on apixaban or rivaroxaban therapy at thepoint of care and could be useful in critical bleeding situations.

Conflict of Interest Statement

DA received a speaker’s fee for lecturing from CSL Behring. SKL re-ceived honoraria for lecturing, travel reimbursement and consultingfees within the last 10 years from Pfizer-BMS, TEM International, and

Table 4LowTF–ROTEM® parameters in the patient groups [mean (±SD)].

Apixaban (n = 3) Rivaroxaban (n = 17) Control (n = 20)

CT (s) 541 (±32)⁎ 865 (±536)⁎ 333 (±72)t,MaxVel (s) 615 (±39) 817 (±276)⁎ 408 (±93)CFT (s) 100 (±6) 261 (±208)⁎ 100 (±35)A10 (mm) 60 (±6) 44 (±18)⁎ 60 (±8)

⁎ = significantly different from the control group (p b 0.05).

Verum Diagnostics. None of the other authors declares any conflict ofinterest.

Acknowledgements

We would like to thank S. Belik for determining the chromogenicantiXa apixaban and rivaroxaban plasma concentrations. This studyhas been funded by the Department of Anaesthesia, General IntensiveCare and Pain Control of the Medical University of Vienna.

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