Summary

• Several reports indicate that XARELTO can result in false positive lupus anticoagulant (LA) test results when using the dilute Russell viper venom test (dRVVT).^1-8
• Treatment with XARELTO has been shown to interfere dose-dependently with one-stage and chromogenic factor VIII activity (FVIII:C) measurements, with lower levels observed.⁹
• One study demonstrated significant decreases in clotting factor activity and prolongation of protein S (PS) activity 2 hours after XARELTO administration, but no effect on protein C activity. Determination of D-dimer, the enzyme-linked immunosorbent assay (ELISA) test for heparin-platelet factor 4 (HPF4) antibodies, detection of anticardiolipin antibodies, and von Willebrand factor (vWF) activity were not influenced by XARELTO.³
• A XARELTO anti-factor Xa activity assay measured with XARELTO calibrators and controls provided a reliable assessment of XARELTO plasma levels in patients receiving anticoagulant therapy.¹⁰
• Results from three studies have demonstrated that, at the concentrations examined, XARELTO had minimal effects on activated protein C resistance (APCR) assays. One of these studies also determined that protein S functional activity assay provided falsely normal results in XARELTO treated patients.^11-13
• In a study of 152 patients taking a factor Xa inhibitor (XARELTO, edoxaban, or apixaban) who were diagnosed with atrial fibrillation (AF) or venous thromboembolism (VTE), XARELTO increased PS activity in a dose-dependent manner (r=0.792, P=0.0001). XARELTO had no effect on PS activity when measured using the chromogenic assay nor did it have any effect on the amount of the PS antigen.¹⁴
• Additional citations identified during a literature search are included in the REFERENCES section for your review.^15-22

CLINICAL STUDIES

Arachchillage et al (2015)¹ sought to establish the influence of therapeutic-dose XARELTO on LA detection, and of antiphospholipid antibodies (aPL) on the anticoagulant action of XARELTO.

• XARELTO and 52 immunoglobulin G (IgG) preparations (20 of which were from LA-positive antiphospholipid syndrome [APS] patients, 12 of which were from LA-negative APS patients, and 20 of which were from normal controls) were spiked into pooled normal plasma for relevant studies. LA detection was studied in APS patients receiving XARELTO 20 mg once daily.
• In vitro spiking of samples with XARELTO showed no false positive LA with Textarin time, Taipan venom time/Ecarin clotting time (TVT/ECT), dilute PT, and in-house dRVVT.
• False positives were seen in the majority of normal controls and LA-negative IgG with 2 commercial dRVVT reagents at 250 ng/mL but not 50 ng/mL XARELTO.
• As part of the ex vivo studies performed, 6 LA-positive patients receiving XARELTO remained LA-positive with TVT/ECT and dRVVT at peak (162-278 ng/mL) and trough (36-80 ng/mL) XARELTO levels.
• Six LA-negative patients became (apparently) LA-positive with 2 dRVVT reagents (test/confirm ratio median [confidence interval], 1.6 [1.3-1.8], 1.6 [1.4-1.9]) but not with TVT/ECT at peak XARELTO levels, and remained LA-negative with both dRVVT reagents and TVT/ECT at trough levels.
• aPL positive IgG spiking of pooled normal plasma had no effect on XARELTO’s anticoagulant action on thrombin generation or XARELTO anti-Xa levels.

Goralczyk et al (2015)² investigated the time-dependent effects of XARELTO with the detection of LA.

• Ten patients treated with XARELTO (20 mg/day) for unprovoked venous thromboembolism (VTE) were referred for thrombophilia screening.
• Thrombophilia screening consisted of antithrombin, protein C, free protein S, factor VIII, factor V Leiden, prothrombin gene G202010A mutation, total homocysteine, LA, anticardiolipin, and anti-β2 glycoprotein I antibodies testing. dRVVT-based assays were used for LA testing.
• Blood samples were drawn between 2 and 6 hours after XARELTO administration. Most lupus anticoagulant screening tests were positive. All but 1 patient was negative for antiphospholipid antibodies.
• Confirmatory tests were performed ≥24 hours after XARELTO administration, yielding negative results.
• Results indicate that to reliably evaluate for lupus anticoagulant, testing should occur at least 24 hours after the last dose of XARELTO to avoid false-positive results.

Mani et al (2013)³ investigated the time-dependent effects of XARELTO on clotting factor activities and thrombophilia screening assays in ex vivo plasma samples from 40 patients.

• Patients were given XARELTO 10 mg once daily for postoperative thrombosis prophylaxis after hip or knee replacement surgery.
• Plasma samples were taken at 2 hours, 12 hours, and 24 hours (immediately prior to the next dose) after XARELTO administration to measure activities of clotting factors II, V, VII, VIII, IX, X, XI, XII, and XIII; protein C and protein S levels; LA; anticardiolipin IgG and IgM; D-dimer, HPF4 antibodies and screening tests for vWF.
• Two hours after XARELTO administration, there were significant decreases in the activities of factor II, V, VII, VIII, IX, X, XI, and XII, with a diminished effect at 12 hours. Dilution of plasma samples resulted in neutralization of these interferences (for factor VIII and factor X activities). The chromogenic assay used to determine factor XIII activity showed no significant differences at different time intervals after XARELTO administration.
• A clotting assay used to determine protein S activity showed a significant prolongation of clotting time and an increase in protein S activity 2 hours after XARELTO administration, while a chromogenic assay determined that there were no significant differences at different time intervals after XARELTO administration for protein C activity.
• The ratio of the dRVVT-LAC assay, which is a factor Xa-based assay, showed a significant increase in LA activity 2 hours after XARELTO administration. In contrast, the MIXCON-LA assay was not significantly influenced by XARELTO.
• Determination of D-dimer, the ELISA test for HPF4 antibodies, detection of anticardiolipin antibodies, and vWF activity were not significantly influenced by XARELTO.

Van Os et al (2011)⁴ evaluated the interference of XARELTO with the detection of LA.  

• Testing was done in normal pooled plasma, LA-positive plasma from systemic lupus erythematosus (SLE) patients, and LA-negative plasma from SLE patients, which were each spiked with XARELTO. LA ratios were determined by measuring the LA-dependent and –independent coagulation times, through the following screening tests: dRVVT screen/dRVVT confirm ratio, activated partial thromboplastin time (aPTT) low and high phospholipids, and taipan venom (LA sensitive) and ecarin (not LA sensitive) times.
• In plasma from healthy individuals, XARELTO prolonged the dRVVT LA ratio (which led to a false positive LA signal), but had no influence on the aPTT LA ratio. In plasma from patients with SLE, XARELTO slightly increased the aPTT LA ratio. The ratio of taipan venom time over ecarin time remained uninfluenced by XARELTO.

Kaplan et al (2010)⁵ assessed the utility of LA testing in 22 patients being treated with XARELTO from the EINSTEIN program.^23,24

• Blood samples were extracted from patients taking XARELTO 20 mg daily within 12 hours of their last dose, but at no defined time interval from the last dose. Testing for a LA was performed using 3 screening assays (kaolin clotting time [KCT], dilute thromboplastin time [DTT], and dRVVT) and 1 confirmatory test (Stago Staclot LATM).  
• The dRVVT was prolonged in 20/22 patients (range: 1.1-3.4, mean: 2.33±1.5 2 standard deviations [2SD]) (normal ratio: 1.0-1.2), and DTT at 1:100 and 1:200 dilutions were prolonged in 13/20 and 16/22 patients (DTT 1:100, range: 1.0-2.0, mean: 1.21±0.45 2SD; DTT 1:200, range: 0.9-2.9, mean: 1.46±0.82 2SD) (normal ratio: 1.0-1.2), respectively. The KCT was normal in 20/22 patients, demonstrating that the KCT was the least sensitive of the LA tests in the presence of XARELTO. The confirmatory test was performed in 13 patients and failed to demonstrate correction in all patients tested.

Merriman et al (2011)⁶ reported in a Letter to the Editor the effect of XARELTO on LA screening tests performed in 21 patients from the EINSTEIN program^23,24

• Nineteen of 21 patients who had LA testing while taking XARELTO had a positive screening test, taken between 1 and 19.5 hours postdose.
• Of all screening tests, the dRVVT showed to be the most abnormal (mean 2.3±0.8, normal range 1.0-1.2) with an elevated/equivocal normalized ratio (=RVV screen ratio/RVV confirm ratio) in 16 of 18 subjects. The DTT was also prolonged (mean value 1.4±0.4, normal range 0.8-1.2), but mostly corrected to normal values with a mix of normal plasma. The KCT was shown to be abnormal in only two of the 21 patients.
• A total of 12 of 19 patients tested negative for the aPTT phospholipid correction test (STAClot method), which normally would have been positive if true LA were detected.
• Repeat testing after discontinuing anticoagulation therapy in 13 of the 19 patients who had abnormal LA testing on XARELTO showed all but 1 tested negative for the presence of LA.
• The authors concluded that XARELTO can cause false positive results on LA testing and therefore should not be performed when patients are taking XARELTO.

Antovic et al (2017)⁸ tested the effects of apixaban, dabigatran, and XARELTO on assays routinely used for diagnosing lupus anticoagulant (LA) in patients treated in real life settings.

• Total of 67 plasma samples were studied from atrial fibrillation patients, apixaban (n=17; 10 mg/day; 5 mg twice/day), dabigatran (n=30; 220 mg/day), and XARELTO (n=20; 10 mg/day).  All samples were not known to have LA.
• Tested samples used diluted Russell’s viper venom time, dRVVT, (LA-screen and LA-confirm, Life Diagnostics) and activated partial thromboplastin time, aPTT, (aPTT-LA, Diagnostica Stago and aPTT-Actin FS, Siemens Healthcare Diagnostics). A BCS XP analyzer (Siemans) was used to perform all analyses.
  • Ratios analysis of dRVVT and aPTT was as follows: ratios of <1.2 were considered negative, ratios >1.4 considered positive, and if 1.2-1.4 LA could not be ruled out.
• Concentrations of apixaban and XARELTO were indirectly quantified by STA^®-Liquid Anti-FXa (Diagnostica Stago), a chromogenic assay. Dabigatran concentration was determined by using the Hemoclot thrombin inhibitors^® (HTI) assay (HYPHEN BioMed).
  • Plasma concentration ranges were as follows: Apixaban, 36-178 µg/l; dabigatran, 8-172 µg/l; and XARELTO, 8-437 µg/l.
• LA diagnosis was negative in 4 (24%) apixaban, 8 (27%) dabigatran and 5 (25%) XARELTO patient samples.
• LA positivity was determined in 7 (41%) apixaban, 5 (17%) dabigatran and 10 (50%) XARELTO patient samples.
• A concentration dependent effect of all 3 drugs on dRVVT-based results was observed. There was no effect on aPTT-LA results at concentrations below 100 µg/l for XARELTO and below 70 µg/l for apixaban. Dabigatran altered aPTT-LA results over the entire concentration range.

Tichelaar et al (2011)⁹ expressed in a Letter to the Editor the effect of XARELTO on FVIII:C measurements in 89 patients from the EINSTEIN program^23,24

• If anticoagulation treatment was indicated for 6 months, blood samples were taken at baseline, at month 3 (t=1), and month 6 (t=2). If anticoagulation treatment was indicated for 12 months or indefinitely, blood samples were taken at baseline, at month 6 (t=1), and month 12 (t=2).
• To confirm results in the EINSTEIN study population, FVIII:C levels from pooled normal plasma spiked with XARELTO in healthy volunteers before and after administration of a XARELTO 20 mg dose were also measured.
• Baseline levels of FVIII:C were not statistically different between patients on XARELTO and vitamin K antagonists (VKA). Mean FVIII:C levels dropped as treatment continued in both groups, with overall levels significantly lower in the XARELTO group. Results are displayed in Table: Results of FVIII:C Measurement In Vivo and In Vitro.
• A dose-dependent interference with XARELTO concentrations higher than 50 ng/mL in the one-stage FVIII:C assay and with XARELTO concentrations higher than 150 ng/mL in the chromogenic assay were observed.

Martinuzzo et al (2013)⁷ investigated the rate of false-positive results of LA tests in patients who previously had negative results that receive dabigatran, XARELTO, or enoxaparin.

• Blood plasma samples were taken from 22 patients taking dabigatran 110 mg twice daily (BID) for atrial fibrillation (AF), 22 patients taking XARELTO 10 mg daily for VTE prevention after elective total hip or knee replacement, 4 patients taking XARELTO 15 mg BID for AF, and 15 patients taking enoxaparin 40 mg daily for the treatment of a VTE or secondary prevention in pregnant women.
• Some of the tests evaluated were prothrombin time (PT), aPTT, dRVVT1 screen, and silica clotting time (SCT).  The prevalence of false-positive results was calculated as the rate of test results above the cutoff values calculated for each screening, mixing, and confirmatory process.
• Patients taking dabigatran BID and XARELTO BID had higher rates of PT, APPT, SCT, and dRVVT prolongation than those taking prophylactic XARELTO once daily. Both XARELTO groups showed negative SCT screen/confirm.  Patients taking enoxaparin had lesser extent of prolongation of aPTT, dRVVT1 screen, and SCT screen as compared to dabigatran and XARELTO BID.
• A high percentage of patients taking XARELTO presented prolonged dRVVT screen without correcting with normal plasma and most of them presented a screen/confirm normalized ratio above the cutoff value.
• XARELTO was found to affect the dRVVT test the most, leading to a high prevalence of false-positive results.

Rathburn et al (2015)¹⁰ assessed the clinically available methods used to analyze patient XARELTO levels.

• The aims of this prospective cohort study were to correlate a chromogenic anti-factor Xa assay using XARELTO Calibrators with serum drug levels via HPLC-MS/MS (XARELTO MS) in patients currently receiving XARELTO, and to correlate the PT/PTT, thrombin generation (Calibrated Automated Thromograph [CAT] assay) and Thromboelastograph (TEG) with anti-factor Xa activity (XARELTO activity) and XARELTO MS.
• A total of 22 adults who were receiving XARELTO (n=8 for AF, n=13 for VTE, and n=1 for peripheral arterial disease; n=3 at 10 mg/day, n=3 at 15 mg/day, and n=16 at 20 mg/day) and 10 healthy controls were enrolled in the study.
• Patients receiving XARELTO had a total of 3 blood draws, the first measured at least 2 hours after ingestion of XARELTO and then subsequent measurements at 2 and 4 hours after initial measurement, for a total of 66 samples. TEG activity was measured at 1 random point for each patient who received XARELTO.
• A strong correlation between XARELTO activity and plasma XARELTO MS (r=0.99) was seen at a concentration range of 25-460 ng/mL.
• A statistically significant correlation (P<0.05) was seen between PT/international normalized ratio (INR) and plasma measurements of XARELTO detected through XARELTO MS (r=0.68) and XARELTO activity (r=0.69).
• The peak height (r=-0.50) and lag time (r=0.57) CAT correlated with XARELTO MS measurements.
• There was no correlation between XARELTO MS and PTT, TEG R (time to clot), TEG MA (clot strength), and endogenous thrombin potential.

Goralczyk et al (2016)¹¹ studied the effects of XARELTO on the Russell viper venom time (RVVT)-based Activated protein C resistance (APC-R) assay in real life patients compared to patients taking vitamin K antagonist (VKA) following venous thromboembolism (VTE) and suspected of thrombophilia.

• Outpatients (N=168) were enrolled with documented VTE suspected of thrombophilia.
  • Eligibility included longer than 3 months since last thrombotic incident, positive family history, VTE at unusual sites, thrombotic incident despite prophylaxis. Patients receiving XARELTO (20 mg/day), acenocoumarol or warfarin were eligible for inclusion.
  • Patients with known malignancy, acute VTE, creatinine clearance (CrCl) <50 ml/min, acute infection, and trauma or surgery in last 12 weeks were excluded.
• The RVVT-based ProC Ac R assay (Siemens) was used to determine APC-R. A chromogenic assay for direct factor Xa inhibitors, Biophen DiXal (Hyphen Biomed) and the Biophen XARELTO Plasma Calibrator set were used to determine XARELTO concentrations. Real-time PCR using the TaqMan genotyping assay (Life Technologics Co.) was used to detect factor V leiden (FVL) mutations.
  • APC-R ratios <1.8 were positive for FVL, values between 1.8-2.9 were equivocal, and >2.9 negative for FVL.
• Of the 168 outpatients, 81 were treated with XARELTO, 54 were treated with VKA and 33 patients were off anticoagulation.
  • APC-R was detected in 23 (28%) of the patients treated with XARELTO. XARELTO concentrations were measured in 56 (69%) of the 81 XARELTO treated patients, their last dose having been taken 2-48 hours prior to blood collection.
  • In the VKA treated patients APC-R was detected in 15 (28%)
  • In the no treatment group APC-R was identified in 4 (12%).
• APC-R ratios in patients treated with XARELTO were similar when compared with the no treatment group, without correlation with XARELTO concentrations (from 0-303 µg/l).
• False-negative or false-positive APC-R ratios were not identified in any of the XARELTO treated patients.

Gessoni et al (2016)¹² assessed ex vivo and in vitro interference of XARELTO on a prothrombinase-based assay for Activated protein C resistance (APCr) detection.

• Fresh plasma samples were obtained from 51 subjects in the ex vivo study.  All 44 subjects with factor V (FV) wild type and 7 with factor V Leiden (FVL) heterozygous were treated with XARELTO.
  • Plasma samples for the FV group had XARELTO concentrations of 0, <100 ng/mL, 100-200 ng/mL, 200-300 ng/ml, or >300 ng/mL, while the FVL group had XARELTO concentrations of 0, <100 ng/mL, 100-200 ng/mL.
• Frozen plasma samples in the in vitro study were obtained from 15 subjects. Six subjects were normal, 6 subjects heterozygous FVL carriers, and 3 homozygous FVL carriers.
  • Each specimen was divided into six aliquots prepared as follows: no XARELTO, or spiked with XARELTO at a final concentration of 50 ng/mL, 100 ng/mL, 200 ng/mL, 300 ng/mL, and 400 ng/mL.
• The concentration of XARELTO was evaluated using an antiFXa assay (Hyphen Biomed) and a Sysmex CA-7000 analyzer (Sysmex Europe).
• APCr was evaluated using Pefakit APC-R FVL, a prothrombinase-based assay, and a Sysmex CA-7000 analyzer.
• Results from both studies, ex vivo and in vitro, showed no significant interference of XARELTO concentration on APCr ratio. This was observed for XARELTO concentrations up to 400 ng/ml in FV wild-type and FVL carriers, both homozygous and heterozygous.

Maryamchik et al (2018)¹³ investigated the magnitude of XARELTO interference in activated protein C resistance (APCR) and protein S activity testing in real world patients.

• Over a 2.5-year period, 4 groups of 15 patients (total n=60) were compared using APCR test results.
  • Group 1: Factor V Leiden (FVL) heterozygous plus XARELTO.
  • Group 2: FVL heterozygous not taking XARELTO.
  • Group 3: FVL-normal (“wild type”) plus XARELTO.
  • Group 4: Normal APCR ratio not taking XARELTO.

During the same period, 32 XARELTO treated patients of the 60 patients underwent protein S testing, both activity assay and free-antigen assay.

• Patient samples were tested using an aPTT-based APCR assay with dilution in factor V-deficient plasma (Coatest APC Resistance V assay, Chromogenix), and an FVL DNA assay (Invader assay, Hologic).  ACPR ratios 2.0 and below were considered abnormal, and values of 2.2 or lower had additional workup by DNA testing to ensure detection of FVL.
• Protein S testing measured functional activity using STACLOT Protein S assay (Stago) and free-antigen using Asserachrom Free Protein S assay (Stago), free-antigen served as a control.
• The concentration of XARELTO was determined by an anti-Xa assay (Stachrom, Stageo) provided in u/mL using a low-molecular weight heparin calibrator and converted to XARELTO concentration using XARELTO calibration curves.
• XARELTO FVL heterozygous patients (Group 1) had lower ACPR ratios than FVL-wild type (Group 3) (P<0.001).  The mean APCR was 1.75 + 0.12 (Group 1) vs 2.63 + 0.23 (Group 3). In FVL heterozygous patients not taking XARELTO (Group 2) and patients taking XARELTO (Group 1) the mean APCR was 1.64 + 0.3 vs 1.75 + 0.12 (P=0.005), respectively. Regardless of XARELTO use, no cases of FVL were missed.
• In the Protein S study, of the 32 patients treated with XARELTO 8 (25%) had FVL and 24 (75%) had wild-type factor V. Mean protein S functional activity was 124.7% + 27.9% and mean free-antigen was 83.3% + 14.8% (P<0.001), the difference statistically significant. Low free-antigen (range, 58%-67%) was seen in 4 of the 32 patients (12.5%) yet functional activity was falsely normal (range, 75%-130%), missing all cases of low protein S if protein S activity testing were used alone in XARELTO patients.

Terakami et al (2022)¹⁴ compared conventional clotting and chromogenic assays to investigate the effect that FXa inhibitors (XARELTO, edoxaban, and apixaban) have on PS activity.

• The study included 152 patients diagnosed with AF or VTE. Patients were placed into two groups: those already on anticoagulant treatment or those newly prescribed FXa inhibitor therapy.
• For patients already on XARELTO, blood samples were collected in tubes containing 3.2% sodium citrate 2 hours after oral administration. Blood samples were collected before as well as 2 and 4–8 weeks post-initiation of treatment in 7 patients that were newly prescribed XARELTO.
  • Clotting assay: Stago STA Staclot Protein S (Fujirebio Inc., Tokyo, Japan), Protein S Ac (Siemens), and HEMOCLOT Protein S (Hyphen BioMed) were used to evaluate PS activity before and after XARELTO was newly introduced.
  • Chromogenic assay: Total Protein S activity “Shino-Test” (Shino-Test Corp., Kanagawa, Japan) was used to measure total PS activity.
• When PS activity was measured using the clotting assay reagents, XARELTO increased PS activity in a dose-dependent manner (r=0.792, P=0.0001). XARELTO had no effect on PS activity when measured using the chromogenic assay nor did it have any effect on the amount of the PS antigen.
• Thrombin-based and Xa-based assays were also performed.
  • XARELTO had no effect on antithrombin (AT) activity when measured using the thrombin-based assay with a chromogenic method.
  • XARELTO increased AT activity in a dose-dependent manner when measured using the Xa-based assay with a chromogenic method (r=0.844, P<0.0001).
• Prothrombin time (PT) and aPTT were evaluated in patients before and after the initiation of XARELTO therapy to assess if the changes in clotting time were associated with the introduction of XARELTO.
  • The analyses showed that PT and aPTT were significantly prolonged in a dose-dependent manner after XARELTO was introduced.
  • In comparison to other FXa inhibitors, the greatest prolongation of aPTT was observed with XARELTO. XARELTO prolonged aPTT to a greater degree than PT (r=0.851, P<0.001).

LITERATURE SEARCH

A literature search of MEDLINE^®, EMBASE^®, BIOSIS Previews^®, and DERWENT^® (and/or other resources, including internal/external databases) pertaining to this topic was conducted on 13 January 2025.