One-year retrospective analysis of anti-FXa apixaban and rivaroxaban levels demonstrates utility for management decisions in various urgent and nonurgent clinical situations.

OBJECTIVES: Quantification of direct oral anticoagulant (DOAC) plasma levels can guide clinical management, but insight into clinical scenarios surrounding DOAC-calibrated anti-FXa assays is limited. METHODS: Apixaban- and rivaroxaban-calibrated chromogenic anti-Xa assays performed over a 1-year period were retrospectively analyzed. Patient demographics, DOAC history, concomitant medications, and renal/liver comorbidities were obtained. Indications for testing and associated clinical actions were reviewed. Machine learning (ML) models predicting clinical actions were evaluated. RESULTS: In total, 371 anti-FXa apixaban and 89 anti-FXa rivaroxaban tests were performed for 259 and 67 patients in recurring urgent (acute bleeding, unplanned procedures) and nonurgent situations, including several scenarios not captured by existing testing recommendations (eg, drug monitoring, recurrent thromboembolic events, bleeding tendency). In urgent settings, andexanet reversal was guided by radiologic and clinical findings over DOAC levels in 14 of 32 instances, while 51% of apixaban patients qualified for nonreversal strategies through the availability of levels. Levels also informed procedure/intervention timing and supported management decisions when DOAC clearance or DOAC target levels were in question. The importance of clinical context was emphasized by exploratory ML models predicting particular clinical actions. CONCLUSIONS: Although clinical situations are complex, DOAC testing facilitates clinical decision-making, including reversal, justifying more widespread implementation of these assays.

Quantitative analysis of direct oral anticoagulant rivaroxaban by terahertz spectroscopy.

Rivaroxaban, as a direct oral anticoagulant, has been widely used in the treatment and prevention of thrombosis disease (TD). However, even if the same dose of rivaroxaban is taken, different pathophysiological characteristics of TD patients determine the differences in plasma concentrations between individuals, leading to the difficulties of dosage selection and plasma concentration control. Conventional rivaroxaban detection methods, including prothrombin time method, anti-Xa assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS), are not widely used in clinical practice due to the limitations of accuracy, speed and cost. Here, we present a simple quantitative detection method for rivaroxaban by terahertz (THz) spectroscopy. Combining density functional theory (DFT) method and THz spectroscopy, the THz absorption peaks of rivaroxaban and the corresponding low-frequency vibrational modes are studied theoretically and experimentally. We find linear relationships between the amplitudes of these characteristic peaks and the concentrations of rivaroxaban. Based on these linear functions, we can analyse the rivaroxaban concentration with a detection time of 1 minute per test and the lowest detection limit of 2 µmol mL-1. As compared to Raman spectroscopy method (its detection limit is about 80 µmol mL-1), our method has more potential and is practical for the clinical quantitative detection of rivaroxaban as well as other direct oral anticoagulants.

Analytical verification and comparison of chromogenic assays for dabigatran, rivaroxaban and apixaban determination on BCSXP and STA Compact Max analysers.

INTRODUCTION: The aim of the study was to perform analytical verification and comparison of chromogenic assays for determination of dabigatran, rivaroxaban and apixaban concentration on BCSXP and STA Compact Max analysers. MATERIALS AND METHODS: Precision, linearity, measurement uncertainty estimation and determination of limit of blank, limit of determination and limit of quantification were calculated. Analytical performance specifications were set according to manufacturer specifications and literature data on between laboratory variability. Comparison of the methods was done using Bland-Altman and Passing-Bablok regression analysis. RESULTS: Obtained results have shown acceptable precision on STA Compact Max only for dabigatran (CV = 3.5%) at lower concentration level comparing to manufacturer declaration (CV = 3.6%). On BCSXP, the highest coefficient of variation has been shown for apixaban (6.1%) at lower concentration level. Within laboratory precision was not met on STA Compact Max for all assays. Bland-Altman analysis has shown statistically significant bias for dabigatran (23.2%, 95%CI 11.2 - 35.3; P < 0.001) and apixaban (8.4%, 95%CI 1.2 - 15.6; P = 0.023). Passing-Bablok regression analysis has shown systematic and proportional deviation between methods for rivaroxaban (y = 6.52 (2.94 to 11.83) + 0.84 (0.80 to 0.89) x. CONCLUSION: Chromogenic assays for dabigatran, rivaroxaban and apixaban on BCSXP and STA Compact Max analysers are shown as methods with satisfactory long-term analytical performance specifications for determination of direct oral anticoagulants in clinical laboratories. However, we cannot recommend interchangeable use because of the significant bias between assays.

Using the PCI-IS Method to Simultaneously Estimate Blood Volume and Quantify Nonvitamin K Antagonist Oral Anticoagulant Concentrations in Dried Blood Spots.

Nonvitamin K antagonist oral anticoagulants (NOACs) have emerged as the preferred choice for the treatment of atrial fibrillation (AF). The establishment of a therapeutic range to minimize bleeding and thrombosis is important for personalized treatment of NOACs. The importance of dried blood spots (DBSs) has increased in medical care. An efficient and effective DBS analytical method could facilitate the concentration management of NOACs. The postcolumn infused internal standard (PCI-IS) method was applied to estimate spot volume and quantify dabigatran, rivaroxaban, and apixaban concentrations on DBS cards. The extraction solvent contented 0.1% formic acid and 70% ACN with a successive extraction procedure. Paired DBS and plasma samples from patients undergoing NOAC therapy (n = 269) were used to calculate conversion factors. [13C6]-Rivaroxaban was selected as the PCI-IS. The quantification accuracy for the three NOACs was within 88.9-104.3%. The RSDs of the repeatability and intermediate precision were below 10%. The obtained conversion factors of DBS to plasma concentrations of dabigatran, apixaban, and rivaroxaban were 1.81, 1.59, and 1.31, respectively. Bland-Altman analysis showed that the % differences between predicted and measured plasma concentrations were within a bias of ±20%. The result showed that PCI-IS was an accurate and efficient LC-MS/MS method to simultaneously estimate blood volume and NOAC concentrations on DBS cards. The stability results revealed that the DBS sampling strategy could improve compound stability. The developed method offers a new strategy for the therapeutic drug monitoring of NOACs and may improve the safe use of these drugs.

Development, validation and application of a new HPLC-DAD method for simultaneous quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma.

Direct oral anticoagulants (DOACs) have been commonly used for the treatment of venous thromboembolism and for the prevention of stroke in patients with atrial fibrillation. Despite not being initially recommended, monitoring DOACs plasma concentrations is now recognized as essential in emergency situations and in special populations. Moreover, the inter-individual variability found in real studies as well as the high reported non-adherence are corroborating the importance of determining the individual relationship between administered doses, plasma concentrations and pharmacological effects. Therefore, accurate but user-friendly bioanalytical techniques are required to monitor DOACs plasma concentrations in routine clinical practice and phase IV clinical trials. Herein, a fast and simple high performance liquid chromatography (HPLC) method coupled to diode array detection (DAD) was developed, validated and applied to quantify the four currently marketed DOACs (apixaban, edoxaban, dabigatran and rivaroxaban). Sample preparation was performed by solid phase extraction followed by evaporation and concentration of the analytes. Chromatographic separation was accomplished within 6 min on a reversed-phase column (octadecyl-silica packing material; 55 mm × 4 mm, 3 µm particle size), applying a mobile phase composed of an aqueous solution of formic acid (0.1 %, v/v) and acetonitrile, pumped with a gradient elution at 30 °C. The proposed method was linear (r2 ≥ 0.993) within the concentration ranges of 0.017-5.28 µg mL-1, 0.066-5.28 µg mL-1, 0.033-5.28 µg mL-1 and 0.017-5.28 µg mL-1 for apixaban, dabigatran, edoxaban and rivaroxaban, respectively, all of them including the expected range of therapeutic concentrations. Overall, intra- and inter-day trueness of quality control samples, including at the lower limit of quantification (LLOQ), varied between -12.98 to 5.79 %, while imprecision was lower than 16.43 %, supporting that the method is accurate and precise in accordance to international guidelines. Recovery and stability were also assessed and allowed the method to be applied in clinical practice, during therapeutic drug monitoring.

Optimization by response surface methodology of a dispersive magnetic solid phase extraction exploiting magnetic graphene nanocomposite coupled with UHPLC-PDA for simultaneous determination of new oral anticoagulants (NAOs) in human plasma.

In this paper a dispersive magnetic-solid phase extraction (MSPE) using a graphene nanocomposite (rG/Fe3O4) followed by ultra high performance liquid chromatography with photodiode array detection has been developed for the simultaneous analysis of new class of oral anticoagulants (NOAs) in human plasma. The performance of the nanocomposite graphene@Fe3O4 on the magnetic solid phase extraction of apixaban, rivaroxaban and dabigatran has been optimized using a Box-Behnken design of experiment. The amount of graphene nanocomposite, the sample pH and the adsorption time were the investigated parameters as a function of the extraction recovery. The analytical method was fully validated based on linearity, limit of detection (LOD), limit of detection (LOQ), inter- and intra-day precision and trueness, and extraction yield. Under optimal condition, excellent linearity (R2 > 0.9987) over the range (0.001-5.0 µg/mL), limit of detection (0.003 µg/mL), precision (0.81-8.97% RSD) and trueness (-5 to 9 % BIAS%) were observed for the target drugs. The average extraction recovery under optimal from plasma samples ranged between 96.6-98.6% for apixaban, rivaroxaban and dabigatran and the internal standard. The proposed method was developed, validated and successfully applied to the measurement of these NOAs in patients. The new approach offers an attractive alternative for the simultaneous analysis of the selected NOAs from plasma samples, providing several advantages including fewer sample preparation steps, ease of performance, and higher recoveries compared to traditional methodologies.

Evaluation of the DOAC-Stop Procedure by LC-MS/MS Assays for Determining the Residual Activity of Dabigatran, Rivaroxaban, and Apixaban.

The effect of direct oral anticoagulants (DOACs) on laboratory tests dependent on the production of their targets, factor IIa and factor Xa (FXa), is a well-known problem and can cause both false positive and negative results. Therefore, the correct interpretation of tests performed in patients receiving DOACs is necessary to avoid misclassification and subsequent clinical consequences. However, even with significant experience, there are situations where it is not possible to assess the influence of some methods. Particularly important is the situation in the diagnosis of lupus anticoagulants using the dilute Russell viper venom timetest, which is based on direct FXa activation. A very promising solution to this situation is offered by the DOAC laboratory balancing procedure DOAC-Stop. For evaluating the effectiveness of this procedure, 60 (20 apixaban, 20 dabigatran, and 20 rivaroxaban) patients treated with DOACs were enrolled. All patient samples were analyzed for the presence of individual DOAC types and subsequently subjected to the DOAC-Stop procedure.We evaluated its effectiveness by our own high-performance liquid chromatography-coupled tandem mass spectrometrymethod, which simultaneously sets all high-sensitivity DOACs. Unlike coagulation tests based on the determination of the residual effects of DOACs on target enzymes, which is complicated by extensive interindividual variation, this methodology is highly specific and sensitive.The DOAC-Stop procedure eliminated dabigatran from 99.5%, rivaroxaban from 97.9%, and apixaban from 97.1% of participants in our group. Residual amounts did not exceed 2.7 ng/mL for dabigatran, 10.9 ng/mL for rivaroxaban, or 13.03 ng/mL for apixaban, which are safe values that do not affect either screening or special coagulation tests.

Use of specific anti-Xa levels in acute kidney injury to transition patients from oral factor Xa inhibitors to i.v. heparin infusion.

PURPOSE: This case series presents 3 patients with acute kidney injury taking apixaban or rivaroxaban and transitioning to a heparin infusion. SUMMARY: Case 1 was a 78-year-old man admitted with respiratory failure, acute decompensated heart failure, and acute kidney injury. He was taking apixaban for atrial flutter. He was transitioned to an i.v. heparin infusion and had 2 consecutive heparin antifactor-Xa levels greater than 2 units/mL. Heparin was held and resumed about 36 hours later when the apixaban anti-Xa level was less than 50 ng/mL. Case 2 was a 55-year-old man admitted with acute kidney injury, taking apixaban for a recent deep vein thrombosis. Apixaban anti-Xa levels were monitored and i.v. heparin was initiated when the level was less than 100 ng/mL, about 56 hours after the last apixaban dose. Case 3 was a 64-year-old woman admitted with sepsis and acute kidney injury taking rivaroxaban for pulmonary embolism, which occurred 2 weeks prior to admission. Rivaroxaban anti-Xa levels were monitored and i.v. heparin was initiated about 36 hours after the last dose when the level was less than 100 ng/mL. The management strategy did not lead to any thrombotic outcomes; however, 1 patient experienced bleeding. CONCLUSION: Specific anti-Xa levels for rivaroxaban and apixaban appeared to be helpful in the transition of 3 patients to unfractionated heparin infusions in the setting of acute kidney injury. These levels provided enhanced, individualized care and likely helped avoid over and under anticoagulation.

Diagnostic and therapeutic approach in adult patients with traumatic brain injury receiving oral anticoagulant therapy: an Austrian interdisciplinary consensus statement.

There is a high degree of uncertainty regarding optimum care of patients with potential or known intake of oral anticoagulants and traumatic brain injury (TBI). Anticoagulation therapy aggravates the risk of intracerebral hemorrhage but, on the other hand, patients take anticoagulants because of an underlying prothrombotic risk, and this could be increased following trauma. Treatment decisions must be taken with due consideration of both these risks. An interdisciplinary group of Austrian experts was convened to develop recommendations for best clinical practice. The aim was to provide pragmatic, clear, and easy-to-follow clinical guidance for coagulation management in adult patients with TBI and potential or known intake of platelet inhibitors, vitamin K antagonists, or non-vitamin K antagonist oral anticoagulants. Diagnosis, coagulation testing, and reversal of anticoagulation were considered as key steps upon presentation. Post-trauma management (prophylaxis for thromboembolism and resumption of long-term anticoagulation therapy) was also explored. The lack of robust evidence on which to base treatment recommendations highlights the need for randomized controlled trials in this setting.

Influence of DOAC Stop on coagulation assays in samples from patients on rivaroxaban or apixaban.

INTRODUCTION: Direct oral anticoagulants (DOACs) require no laboratory monitoring, but they interfere with almost all clotting tests to a varying degree, depending on the DOAC, assay principles and reagents used. DOAC Stop (Haematex Research, Sydney, Australia) has recently been shown to adsorb DOACs from spiked and patient plasmas. The aim of our work was to investigate the DOAC Stop effect on a range of haemostasis assays on plasmas collected from patients on rivaroxaban or apixaban, to see whether it removes the effect of these drugs to enable more accurate interpretation of coagulation assays. METHODS: Samples from patients anticoagulated with either rivaroxaban, apixaban or no anticoagulant were tested for prothrombin time (PT), activated partial thromboplastin time (APTT), DOAC-specific anti-Xa assay, factor VIII (one-stage and chromogenic assay) and DRVVT (low and high phospholipid) before and after sample treatment with DOAC-Stop. RESULTS: DOAC Stop significantly removed the effects of rivaroxaban and apixaban on PT, APTT, anti-Xa activity, factor VIII (one-stage and chromogenic assays), and DRVVT (low and high phospholipid reagents), and reduced the number of false positive of lupus anticoagulant interpretations in patients on rivaroxaban. There was no effect on the results from patients that were not anticoagulated. CONCLUSION: This small study suggests that it is likely that DOAC Stop can be used in laboratories to screen for coagulopathy or lupus anticoagulants in samples containing rivaroxaban or apixaban. Care should be taken in the interpretation of results since complete reversal of the anti-Xa effect did not occur in every sample.

Quantitative Assessment of Poorly Soluble Anticoagulant Rivaroxaban by Microemulsion Electrokinetic Chromatography.

Microemulsion electrokinetic chromatography (MEEKC) is an electrophoretic methodology based on the separation of compounds by a microemulsionated electrolyte. There are few options for the evaluation of the stability and content of the oral anticoagulant rivaroxaban (RIV) in pharmaceutical formulations. RIV has low water solubility and undergoes ionization only under restricted pH conditions (pH < 1 or pH > 13), thus, hindering the application of free zone capillary electrophoresis as an analytical method. Therefore, the work aimed at developing and validating a stability-indicating MEEKC method for the analysis of RIV in pharmaceutical formulations. Separation was performed in a fused-silica capillary applying a voltage of 30 kV. The microemulsion system consisted of 13 mM tetraborate, pH 9.75 + 1.2% SDS + 1.0% ethyl acetate + 2.4% butanol. The linearity range was 25-150 µg mL-1, with r = 0.9982. Drug degradations were performed in acid and basic media (HCl 1 M and NaOH 0.1 M, respectively), oxidation with 3%H2O2, 60°C temperature and exposure to UV-C radiation. No interferences with RIV or internal standard peaks were detected. Method robustness was accessed through Plackett-Burman experimental design, after evaluation of model validity. Trueness values between 100.49 and 100.68% were obtained with repeatability. The method developed was found appropriate for quality control of RIV tablets, as a consistent analytical technique that is considered less damaging to the environment due to its low consumption of organic reagents.

International Council for Standardization in Haematology (ICSH) Recommendations for Laboratory Measurement of Direct Oral Anticoagulants.

This guidance document was prepared on behalf of the International Council for Standardization in Haematology (ICSH) for providing haemostasis-related guidance documents for clinical laboratories. This inaugural coagulation ICSH document was developed by an ad hoc committee, comprised of international clinical and laboratory direct acting oral anticoagulant (DOAC) experts. The committee developed consensus recommendations for laboratory measurement of DOACs (dabigatran, rivaroxaban, apixaban and edoxaban), which would be germane for laboratories assessing DOAC anticoagulation. This guidance document addresses all phases of laboratory DOAC measurements, including pre-analytical (e.g. preferred time sample collection, preferred sample type, sample stability), analytical (gold standard method, screening and quantifying methods) and post analytical (e.g. reporting units, quality assurance). The committee addressed the use and limitations of screening tests such as prothrombin time, activated partial thromboplastin time as well as viscoelastic measurements of clotting blood and point of care methods. Additionally, the committee provided recommendations for the proper validation or verification of performance of laboratory assays prior to implementation for clinical use, and external quality assurance to provide continuous assessment of testing and reporting method.

Dual-target inhibitor screening against thrombin and factor Xa simultaneously by mass spectrometry.

An accurate, rapid, and cost-effective methodology for enzyme assay is highly demanded to screen the effect of compounds on target at the molecular level. Thrombin (EC 3.4.21.5) and factor Xa (FXa, EC 3.4.21.6) have been identified as the critical targets for the development of potential drugs with anticoagulant activity. In this study, a rapid, sensitive and accurate assay based on UHPLC-MS/MS method has been developed for inhibitor screening against thrombin and factor Xa simultaneously. For thrombin and factor Xa, the Michaelis-Menten constants (Km) were calculated to be 6.14 and 57.27 µM, respectively. The inhibition constants (Ki) for two known inhibitors, argatroban and rivaroxaban, were determined to be 16.23 and 0.41 nM, respectively. The assay was further validated through the determination of a high Z' factor value of 0.89. Finally, the developed assay was applied to screen a chemical library against two enzymes. Three hit compounds belonging to a class of sulfated polysaccharides were identified and their targets of inhibition action were further evaluated. The results indicated that the dual-target assay by UHPLC-MS/MS analysis could be used as a reliable method for screening anticoagulant agents.

Point-of-care testing for emergency assessment of coagulation in patients treated with direct oral anticoagulants.

BACKGROUND: Point-of-care testing (POCT) of coagulation has been proven to be of great value in accelerating emergency treatment. Specific POCT for direct oral anticoagulants (DOAC) is not available, but the effects of DOAC on established POCT have been described. We aimed to determine the diagnostic accuracy of Hemochron® Signature coagulation POCT to qualitatively rule out relevant concentrations of apixaban, rivaroxaban, and dabigatran in real-life patients. METHODS: We enrolled 68 patients receiving apixaban, rivaroxaban, or dabigatran and obtained blood samples at six pre-specified time points. Coagulation testing was performed using prothrombin time/international normalized ratio (PT/INR), activated partial thromboplastin time (aPTT), and activated clotting time (ACT+ and ACT-low range) POCT cards. For comparison, laboratory-based assays of diluted thrombin time (Hemoclot) and anti-Xa activity were conducted. DOAC concentrations were determined by liquid chromatography-tandem mass spectrometry. RESULTS: Four hundred and three samples were collected. POCT results of PT/INR and ACT+ correlated with both rivaroxaban and dabigatran concentrations. Insufficient correlation was found for apixaban. Rivaroxaban concentrations at <30 and <100 ng/mL were detected with >95% specificity at PT/INR POCT ≤1.0 and ≤1.1 and ACT+ POCT ≤120 and ≤130 s. Dabigatran concentrations at <30 and <50 ng/mL were detected with >95% specificity at PT/INR POCT ≤1.1 and ≤1.2 and ACT+ POCT ≤100 s. CONCLUSIONS: Hemochron® Signature POCT can be a fast and reliable alternative for guiding emergency treatment during rivaroxaban and dabigatran therapy. It allows the rapid identification of a relevant fraction of patients that can be treated immediately without the need to await the results of much slower laboratory-based coagulation tests. TRIAL REGISTRATION: Unique identifier, NCT02371070 . Retrospectively registered on 18 February 2015.

Measurement of dabigatran, rivaroxaban and apixaban in samples of plasma, serum and urine, under real life conditions. An international study.

BACKGROUND: The utility of measuring non-vitamin K antagonist oral anticoagulants (NOACs) in plasma, serum and urine samples and with the point-of-care test (POCT) on urine samples should be analysed in an international laboratory study. METHODS: The study was performed to determine the inter-laboratory variance of data from two chromogenic assays each for the NOACs rivaroxaban, apixaban and dabigatran, and to analyse the sensitivity and specificity of the POCT assays for factor Xa- and thrombin inhibitors. Plasma, serum and urine samples were taken from six patients in each group on treatment with a NOAC. RESULTS: The inter-laboratory variances, which can be identified best by the coefficient of variation, ranged from 46% to 59% for apixaban, 63% to 73% for rivaroxaban and 39% to 104% for dabigatran using plasma, serum or urine samples and two chromogenic assays for each NOAC. The concentrations were about 20% higher in serum compared to plasma samples for apixaban and rivaroxaban, and 60% lower for dabigatran. The concentration in urine samples was five-fold (apixaban), 15-fold (rivaroxaban) and 50-fold (dabigatran) higher. Sensitivity and specificity of POCT for apixaban, rivaroxaban, and dabigatran were all >94%. CONCLUSIONS: The inter-laboratory study showed the feasibility of measurement of apixaban, rivaroxaban, and dabigatran in plasma, serum and urine samples of patients on treatment. Dabigatran was determined at far lower levels in serum compared to plasma samples. Concentrations of NOACs in urine were much higher compared to plasma. The POCT was highly sensitive and specific for all three NOACs.