Multicenter Evaluation of a Rivaroxaban-Calibrated Anti- Xa Assay against LC-MS/MS in China: Correlation, Accuracy, and Clinical Utility

Multicenter Evaluation of a Rivaroxaban-Calibrated Anti- Xa Assay against LC-MS/MS in China: Correlation, Accuracy, and Clinical Utility | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Multicenter Evaluation of a Rivaroxaban-Calibrated Anti- Xa Assay against LC-MS/MS in China: Correlation, Accuracy, and Clinical Utility Juntao Zhao, Wenhao Cui, Yuying Shi, Jingfeng Tong, Yujing Yang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6989437/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Backgrounds: Rivaroxaban, a direct oral anticoagulant (DOAC) targeting Factor Xa, is widely used without routine monitoring. However, the absence of reliable drug level measurement may hinder clinical decision-making. The study evaluated the correlation between a commercial rivaroxaban-calibrated anti-Xa assay (Riva anti-Xa) and plasma drug levels measured by HPLC-MS/MS (Riva LC-MS), and assess its diagnostic accuracy for clinically relevant thresholds. Methods A spiking experiment and a prospective multi-center study were conducted, using Riva LC-MS as the reference. Correlation and agreement were assessed via Spearman analysis, Bland-Altman plots, and Deming regression. Sensitivity and specificity were calculated of 30, 50, and 100 ng/mL. Results The Riva anti-Xa assay showed excellent correlation with Riva LC-MS (Spearman r = 0.982, 95% CI: 0.970–0.988) and maintained acceptable bias in the presence of interfering substances. Clinical samples showed high concordance with LC-MS results. For concentrations ≥ 30, 50, and 100 ng/mL, the assay yielded sensitivities of 100.0%, 100.0%, and 92.74%, and specificities of 99.58%, 99.49%, and 99.30%, respectively. ROC-AUC values were 0.99 across all thresholds. Conclusion The Riva anti-Xa assay is a reliable, rapid alternative for estimating rivaroxaban levels, with strong correlation to LC-MS and excellent diagnostic accuracy, supporting its use in urgent clinical settings. Health sciences/Biomarkers Health sciences/Diseases Health sciences/Medical research Rivaroxaban anti-Xa assay LC-MS correlation interference resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Rivaroxaban (Riva) is a direct oral anticoagulant (DOAC) that specifically inhibits both free and bound factor Xa activity, thereby blocking thrombin generation and preventing hemostasis. It has been approved for various clinical applications, including the prevention of venous thromboembolism (VTE) following hip or knee replacement surgery, the prevention of cardioembolic stroke in patients with non-valvular atrial fibrillation (NVAF), and the treatment of VTE, including pulmonary embolism (PE) and deep vein thrombosis (DVT) 1 – 3 . Numerous studies have demonstrated that DOACs, including rivaroxaban, outperform warfarin, and multiple professional associations recommend DOACs over warfarin for reducing stroke risk in patients with various indications 4 – 6 . In terms of overall healthcare costs, rivaroxaban has also shown significant savings compared to warfarin 7 . Although rivaroxaban exhibits clear and predictable pharmacological anticoagulant effects with fewer food and drug interactions compared to warfarin and does not require routine coagulation monitoring, understanding its plasma drug concentration is crucial in specific clinical scenarios. These scenarios include perioperative management, elderly patients, non-adherence and non-persistence, those with hepatic or renal impairment, planned thrombolysis, or major bleeding events 8 – 10 . Additionally, real-world cohort studies have demonstrated substantial inter-individual variability in the plasma concentrations of rivaroxaban, influenced by factors such as renal function, age 11 . Furthermore, recent investigations have also highlighted potential interactions with commonly prescribed clinical medications, such as levetiracetam and valproic acid, which may affect rivaroxaban’s pharmacokinetics and anticoagulant efficacy 12 , 13 . Rapid methods for detecting drug concentrations or factor Xa activity levels are essential for monitoring adherence, addressing potential treatment failure, ensuring the safety of perioperative anticoagulation management, and providing optimal care for long-term anticoagulated patients. Although prothrombin time (PT) can serve as a surrogate marker for rivaroxaban efficacy, its correlation is suboptimal 14 , 15 . High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS, LC-MS) can accurately measure rivaroxaban concentrations within the range of 0.50 to 500 µg/L and is considered the gold standard for drug concentration assessment. However, its clinical utility is limited by slow turnaround times and restricted availability 16 – 18 . Anti-factor Xa (anti-Xa) activity assays address these limitations and present a viable option for clinical laboratories. Validated anti-Xa assays have been shown to reliably measure rivaroxaban concentrations 19 , 20 . The objectives of this study are: (1) to investigate the correlation between a commercial rivaroxaban anti-factor Xa assay (Riva anti-Xa, Zybio) and plasma drug concentrations measured by high-performance liquid chromatography-tandem mass spectrometry (Riva LC-MS); (2) to assess the interference resistance of the Riva anti-Xa assay by evaluating its performance in plasma samples spiked with potential interfering substances as well as clinical samples with or without interferents; and (3) to evaluate the diagnostic capability of the Riva anti-Xa assay in accurately predicting clinically relevant decision-making thresholds in practice. Materials and Methods Spiking experiments Standardized human plasma was spiked with varying concentrations of rivaroxaban reference standards. Rivaroxaban stock solutions were prepared in dimethyl sulfoxide (DMSO) and diluted to obtain eight final concentrations ranging from 20 to 520 ng/mL. The rivaroxaban anti-Xa activity assay was performed using a fully automated coagulation analyzer, EXT4800 (Zybio, Inc.). Interference resistance evaluation experiment This study evaluated the interference resistance of the Riva anti-Xa activity assay performed on the EXT4800 coagulation analyzer. The potential interfering substances included bilirubin, hemoglobin, triglycerides, dabigatran, unfractionated heparin (UFH), and low-molecular-weight heparin (LMWH). Each substance was added to standardized human plasma to achieve the following final concentrations: bilirubin at 0, 0.1, 0.2, and 0.3 mg/mL; hemoglobin at 0, 1.0, 1.5, and 2.0 mg/mL; triglycerides at 0, 5, 10, and 12 mg/mL; dabigatran at 0, 50, 250, and 500 ng/mL; UFH at 0, 1.0, and 2.0 IU/mL; and LMWH at 0, 2.0, and 4.0 IU/mL. For each level of interfering substance, low, medium, and high concentrations of rivaroxaban were added (final concentrations of 50, 250, and 400 ng/mL, respectively). Each prepared sample was measured three times using the Riva anti-Xa activity assay. Samples without added interferents served as controls, and the relative bias of each interferent sample was calculated using the formula: Relative Bias (%) = (Mean of Interferent Group) - (Mean of Control Group)/(Mean of Control Group) ×100%. Study population This was a prospective multicenter clinical study that recruited patients on long-term rivaroxaban therapy in clinical practice between March, 2024 and September, 2024 in three Chinese tertiary hospitals. The flow chart of the patients is given in Fig. 1 . All included patients provided written informed consent for study participation. Inclusion criteria were: age ≥ 18 years, current use of rivaroxaban (regardless of indication), and the ability to provide informed consent. Exclusion criteria included: pregnancy, age < 18 years, incomplete sample information, and inability to provide informed consent. Data collection encompassed patient age, gender, indication for rivaroxaban, dosage and duration of rivaroxaban use, interference as well as other concomitant medications. Sample size This clinical study was designed as a methodological comparison and used correlation coefficient testing (for continuous variables) to calculate the sample size. Following the CLSI EP9-A3 guidelines established by the Clinical and Laboratory Standards Institute (CLSI), the correlation coefficient between the test reagent results and the reference method should not be lower than 0.975. Based on the anticipated correlation coefficient of ≥ 0.983 between the Riva anti-Xa assay and the Riva LC-MS results, the parameters were set as follows: α = 0.025 (one-sided), s = 1, β = 0.2, P0 = 0.975, and P1 = 0.983. Using the sample size estimation formula for correlation coefficient testing (continuous variables) and calculated with PASS2021 software, the required sample size was determined to be 210 cases. Considering a 5% exclusion rate and the distribution of samples across three study centers, the final sample size for this clinical trial was set at 267 cases. Sample collection and processing Each enrolled patient underwent a single blood draw. Using sterile techniques, one tube of blood was collected within 24 hours after drug administration in a 3.2% sodium citrate (blue-top) tube. Plasma was separated by standard clinical centrifugation methods, divided into two equal aliquots, and stored at -20°C. All samples were batch-tested at the end of the study. Anti-Xa activity assay A commercial Riva anti-Xa activity assay (Zybio) was employed using rivaroxaban calibrators and quality control materials. After rapid thawing, samples were gently mixed at 37°C. Patient plasma was pre-diluted (1:2, 100 µl) and mixed with a chromogenic substrate solution (250 µl). After incubation at 37°C, factor Xa (250 µl) was added. The reaction was terminated after 120 seconds, and absorbance was measured at 405 nm. All procedures were performed strictly according to the manufacturer's instructions. Quantification of rivaroxaban by LC-MS Rivaroxaban concentrations were quantified using LC-MS technology. To 50 µl of plasma, 10 µl of acetonitrile: water (1:1, v/v), 25 µl of extraction buffer, and 240 µl of precipitation reagent containing an internal standard were added to precipitate proteins and extract analytes. The mixture was centrifuged at 14,000 g for 4 minutes at 20°C. A 20 µl aliquot of the supernatant was diluted to 80 µl with water: methanol (8:2, v/v) and stored at 10°C until analysis. Calibrators and quality control samples were prepared using mixed plasma. Analysis was performed using reverse-phase chromatography on a triple quadrupole mass spectrometer (ABS4500 model). Rivaroxaban was separated by gradient elution at a flow rate of 0.4 ml/min, with the mobile phase consisting of 0.1% formic acid in water (A) and methanol (B). Statistical analysis The baseline characteristics and detection results of the enrolled patients were compiled into an Excel spreadsheet. Categorical variables were presented as case numbers and percentages, while continuous variables were summarized as medians with their 95% confidence intervals. Group comparisons were conducted using the Mann-Whitney U test. The relationship between Riva anti-Xa activity and Riva LC-MS was determined using Spearman correlation analysis. Bland-Altman plots were utilized to evaluate the agreement between Riva anti-Xa activity quantification and Riva LC-MS. Additionally, Deming regression was performed to assess the relationship between the two methods. The receiver operating characteristic curve (ROC) was plotted and the area under the curve (AUC), cut-off, sensitivity and specificity were calculated for clinically relevant drug concentrations of 30, 50, and 100 ng/mL 17 , 21 . All statistical analyses and visualizations were conducted using GraphPad Prism version 10.1.2, with statistical significance set at p < 0.05. Ethics statement The study protocol was approved by the Ethics Committees and institutional review boards of Shanghai Jiaotong University School of Medicine affiliated Ruijin Hospital (No: 2023 − 117), Guangdong Provincial People’s Hospital (No: QX2023-008-01) and The Second Affiliated Hospital of Guangzhou Medical University (No: Q2023-07-01). This study was conducted in accordance with the principles of the Declaration of Helsinki of the World Medical Association and with Good Clinical Practice Guidelines. Results Spiking experiments A spiking experiment was conducted to evaluate the accuracy of the Riva anti-Xa activity assay. As shown in Figure S1 , a strong linear relationship was observed between the actual concentrations of the spiked samples and those measured by the assay. The regression equation yielded a slope of 0.95 (95% CI: 0.86, 1.05), with a Spearman correlation coefficient of 1.0 between the two, indicating a high level of agreement. Basic information of the enrolled participants As shown in the flowchart (Fig. 1 ), we prospectively recruited patients on long-term rivaroxaban therapy who visited the three hospitals between March and September 2024. A total of 267 cases satisfying the inclusion and exclusion criteria were enrolled in this study with a median age of 61 years (95% CI: 59–64), and 60.7% (162/267) were male. The demographic characteristics of the enrolled patients are summarized in Table 1 . Among these patients, five were receiving concomitant heparin therapy at the time of sample collection. The majority of patients (47.2%, 126/267) were prescribed rivaroxaban for venous thromboembolism (VTE), while 28.1% (75/267) were being treated for atrial fibrillation (AF). Table 1 Basic information of the study population Features Patients (n = 267) Age Median (95% CI) 61 (59, 64) Gender (n, %) Male 162 (60.7) Female 105 (39.3) Indication (n, %) Atrial fibrillation 75 (28.1) Venous thromboembolism 126 (47.2) Others 66 (24.7) Rivaroxaban dose (n, %) 2.5 mg 19 (7.1) 5 mg 6 (2.3) 10 mg 124 (46.4) 15 mg 98 (36.7) 20 mg 20 (7.5) Interference (n, %) Yes 43 (16.1) No 224 (83.9) Accuracy of Rivaroxaban anti-Xa activity measurements Overall correlation between Riva anti-Xa activity results and rivaroxaban plasma concentration as determined by HPLC-MS (Riva LC-MS) was strong with a Spearmen’s correlation coefficient of 0.997 (95% CI, 0.996, 0.997) (Fig. 2 , Table 2 ). However, correlation was considerably lower at rivaroxaban plasma concentrations above 200 ng/mL (Table 2 ). The overall bias of the Bland-Altman difference plot is 0.405 with 95% agreement limit from − 16.2 to 17.0 ng/mL (Fig. 3 ). Stratified analysis revealed a trend of increasing bias at higher rivaroxaban plasma concentrations (Table 2 ). Table 2 Accuracy of Riva anti-Xa activity measurements if compared with Riva LC-MS Riva anti-Xa 200 ng/mL n = 62 Total n = 267 Spearman’s correlation coefficient (95% CI) 0.982 (0.970, 0.988) 0.988 (0.983, 0.991) 0.970 (0.951, 0.983) 0.997 (0.996, 0.997) Deming regression Slope (95% CI) 0.997 (0.953, 1.040) 1.03 (0.999, 1.06) 1.03 (0.98, 1.08) 1.003 (0.989, 1.018) Y-intercept (95% CI) 0.280 (-0.815, 1.376) -2.28 (-5.30, 0.74) -9.46 (-23.73, 4.82) -0.015 (-1.379, 1.350) Bland-Altman difference plot Bias (SD) 0.178 (2.16) 0.949 (6.88) -0.51 (14.2) 0.41 (8.45) Lower limit of agreement -4.06 -12.5 -28.3 -16.2 Upper limit of agreement 4.41 14.4 27.3 17.0 Analysis of Interference Resistance The ability to resist interference varies across different reagents and instrument platforms. The EXT4800 coagulation analyzer, as a hybrid system combining optical and magnetic detection technologies, offers both the sensitivity and precision of optical methods as well as the robust interference resistance of mechanical methods. In this study, we assessed the accuracy of the Riva anti-Xa assay in measuring rivaroxaban concentrations in interference-affected samples using the EXT4800 platform. We prepared samples containing varying concentrations of bilirubin, hemoglobin, triglycerides, dabigatran, unfractionated heparin (UFH), and low-molecular-weight heparin (LMWH), and spiked each with low, medium, and high concentrations of rivaroxaban (50 ng/mL, 250 ng/mL, and 400 ng/mL, respectively). Rivaroxaban concentrations were measured using the Riva anti-Xa activity assay, with samples without interferents serving as controls. The relative bias of measurements in the presence of each interferent was calculated accordingly. As shown in the Fig. 4 , the relative bias of rivaroxaban detection using the Riva anti-Xa activity assay remained within ± 10% for bilirubin concentrations up to 0.3 mg/mL, with most biases within ± 5% (Fig. 4 A). For hemoglobin concentrations up to 2.0 mg/mL, the relative bias remained within ± 3% (Fig. 4 B). Similarly, for triglyceride concentrations up to 12 mg/mL, the relative bias was within ± 4% (Fig. 4 C). At a dabigatran concentration of 500 ng/mL, the majority of the relative biases were within ± 3.0%, except for a single measurement at the medium rivaroxaban concentration, which showed a bias of 9.4% (Fig. 4 D). For UFH concentrations up to 2.0 IU/mL, the relative bias was within ± 2% (Fig. 4 E), and for LMWH concentrations up to 4.0 IU/mL, the relative bias was within ± 3% (Fig. 4 F). We further analyzed the batch of clinical samples, among which 43 samples were identified as having potential interferences, including hemolysis, jaundice, lipemia, or concomitant use of other anticoagulants. The differences between rivaroxaban concentrations measured by the Riva anti-Xa activity assay and Riva LC-MS in the interfering and non-interfering samples were compared. No significant differences were observed between the two groups ( Figure S2 ). The correlation between the two detection methods remained high for both interfering and non-interfering samples, with correlation coefficients of 0.994 (95% CI: 0.989–0.997) and 0.997 (95% CI: 0.996–0.998), respectively (Table 3 ). These results indicate that the Riva anti-Xa activity assay on the EXT4800 coagulation analyzer demonstrates strong resistance to interference when measuring rivaroxaban concentrations. Table 3 The influence of interference on the accuracy of Riva anti-Xa activity measurements if compared with Riva LC-MS. With interference, n = 43 Without interference, n = 224 Pearson’s correlation coefficient (95% CI) 0.994 (0.989, 0.997) 0.997 (0.996, 0.998) Deming regression Slope (95% CI) 1.01 (0.985, 1.030) 1.00 (0.987, 1.020) Y-intercept (95% CI) 0.011 (-2.55, 2.57) -0.051 (-1.53, 1.43) Bland-Altman difference plot Bias (SD) 0.944 (6.60) 0.302 (8.77) Lower limit of agreement -12.0 -16.9 Upper limit of agreement 13.9 17.5 Diagnostic performance of the Riva anti-Xa activity assay for predicting clinically relevant decision thresholds Figure 5 illustrates the diagnostic accuracy of the Riva anti-Xa activity assay in identifying clinically relevant rivaroxaban concentrations. For a drug concentration threshold of ≥ 30 ng/mL, the assay demonstrated a sensitivity of 100.0% (95% CI: 88.97–100.0%) with an optimal cutoff value of 29.45 ng/mL, and a specificity of 99.58% (95% CI: 97.64–99.98%). At a threshold of ≥ 50 ng/mL, the sensitivity remained 100.0% (95% CI: 94.87–100.0%) with a cutoff value of 49.70 ng/mL, and the specificity was 99.49% (95% CI: 97.17–99.97%). For a threshold of ≥ 100 ng/mL, the sensitivity was 92.74% (95% CI: 86.78–96.13%) with a cutoff value of 95.70 ng/mL, and the specificity reached 99.30% (95% CI: 96.15–99.96%). The area under the receiver operating characteristic (ROC) curve (AUC) for all three thresholds was 0.99, indicating excellent diagnostic performance of the Riva anti-Xa activity assay in distinguishing clinically relevant rivaroxaban concentrations. Discussion Although rivaroxaban, a direct oral anticoagulant (DOAC), was introduced in China as early as 2009, a simple and practical method for measuring its plasma concentration in routine clinical laboratories has remained unavailable. While such assays are well-established internationally, their implementation in China has been limited. The Riva anti-Xa assay evaluated in this study aims to bridge this gap. To our knowledge, this is the first study to assess the accuracy, diagnostic performance, and interference resistance of this commercial assay in a Chinese clinical setting. As the number of patients using DOACs continues to rise, assessing medication adherence is becoming increasingly important. In other clinical scenarios, such as perioperative evaluations for major or critical organ surgeries, renal dysfunction, extreme body weight, and advanced age, therapeutic drug monitoring is essential to optimize anticoagulation therapy and minimize adverse events 22 – 24 . A case report from the Mayo Clinic described an elderly patient with an abnormally prolonged half-life of apixaban, emphasizing the potential impact of patient-specific factors on drug metabolism and clearance 25 . Similarly, Wu et al. analyzed a cohort of nearly 300 patients receiving rivaroxaban at Hebei Provincial People’s Hospital and reported an association between higher trough concentrations and an increased risk of bleeding events 26 , further highlighting the potential value of continuous monitoring of anti-Xa levels in guiding anticoagulation management. Our study evaluated the accuracy and clinical diagnostic performance of the first Riva anti-Xa assay developed in China. The results demonstrated a high concordance with the reference method, underscoring its reliability and potential utility in routine clinical practice. These findings suggest that Riva anti-Xa assay could serve as a valuable tool for individualized anticoagulation management, particularly in patients with complex clinical conditions. In clinical practice, particularly before invasive procedures or surgeries, it is essential to discontinue anticoagulants. For patients undergoing procedures with a high risk of bleeding, determining the concentration of direct oral anticoagulants (DOACs) is a critical factor in guiding physician interventions. In patients requiring urgent high-risk interventions or experiencing life-threatening uncontrolled bleeding, a DOAC concentration of 50 ng/mL serves as the threshold for deciding whether to administer a DOAC reversal agent. Concentrations above this level necessitate the use of a reversal agent. Furthermore, in all scenarios, including elective surgeries, urgent interventions, or invasive procedures, DOAC concentrations must be below 30 ng/mL to minimize bleeding risk. For thrombolytic therapy consideration, a threshold of 100 ng/mL is critical. This study evaluated the diagnostic performance of the Riva anti-Xa activity assay at these three key clinical decision levels. Results demonstrated that the Riva anti-Xa activity assay exhibits high diagnostic performance in assessing concentrations at these clinically relevant thresholds, confirming its utility in clinical practice. Sample quality is critical for the analytical accuracy of laboratory diagnostics and patient safety. The most common pre-analytical interferences in coagulation laboratories involve hemolysis, jaundice, and lipemia in blood samples. While optical coagulation methods are highly sensitive and accurate, they are less robust against such interferences. Conversely, magnetic bead-based mechanical coagulation methods exhibit strong anti-interference capabilities but are less sensitive and accurate compared to optical methods. The interference resistance varies across different coagulation platforms. Hedeland et al. investigated the impact of hemolysis on 10 coagulation assays using the Stago STA R Max 2 analyzer and reported that hemolysis significantly interfered with anti-Xa activity measurement, with a hemoglobin concentration as low as 0.5 mg/mL leading to a 10% reduction in assay results 27 . In the present study, we employed the EXT4800 optical-magnetic integrated coagulation analyzer, which is designed to exhibit strong resistance to interference 28 . Our findings further confirmed its robustness, as even at a hemoglobin concentration of 2.0 mg/mL, the detection of Rivaroxaban anti-Xa activity remained unaffected in hemolyzed samples (Fig. 5 B). This demonstrates that the EXT4800 analyzer not only maintains reliable performance under challenging preanalytical conditions but also offers a potential advantage for routine coagulation testing in clinical laboratories. This study has several limitations that should be acknowledged. First, although the study included a robust sample size, it was limited to three centers, which may not fully represent the variability in sample handling and patient populations across different institutions. Second, while we assessed interference from hemolysis, jaundice, and lipemia, other potential pre-analytical and analytical factors, such as extreme pH or storage conditions, were not comprehensively examined. Additionally, our study did not include pediatric patients or those with rare coagulation disorders, which limits the generalizability of our findings to these populations. Finally, while the Riva anti-Xa assay demonstrated strong correlation and diagnostic performance against LC-MS, further studies are needed to evaluate its long-term clinical utility and cost-effectiveness in guiding anticoagulation management across diverse clinical scenarios. Conclusion In summary, this study demonstrated that the Riva anti-Xa activity assay provides a highly accurate and reliable method for estimating rivaroxaban plasma concentrations, showing an excellent correlation with the gold-standard LC-MS method. The assay exhibited high sensitivity and specificity in identifying clinically relevant decision thresholds (≥ 30, 50, and 100 ng/mL), with ROC-AUC values of 0.99 across all thresholds. These findings support the Riva anti-Xa assay as a practical, efficient, and accessible tool for clinical laboratories, particularly in scenarios requiring rapid anticoagulation monitoring, such as perioperative management, high-risk bleeding events, and thrombolysis planning. Its simplicity and accuracy make it a valuable alternative to LC-MS for guiding rivaroxaban therapy in real-world clinical practice. Declarations Competing interests The authors have no competing interests to declare. The funding had no involvement in the study design, data collection, analysis, interpretation, or writing of the manuscript, and does not constitute a conflict of interest. Ethics declarations The study protocol was approved by the Ethics Committees and institutional review boards of Shanghai Jiaotong University School of Medicine affiliated Ruijin Hospital (No: 2023 − 117), Guangdong Provincial People’s Hospital (No: QX2023-008-01) and The Second Affiliated Hospital of Guangzhou Medical University (No: Q2023-07-01). Funding This research was funded by Guangzhou Science and Technology Project, grant number 2024A04J3705. Author Contribution Conceptualization, G.L. and Y.F.; Data curation, J.D., J.Z., W.C.; Formal analysis, Y.S.; Funding acquisition, B.Y.; Investigation, Y.Y.; Methodology, J.T., J.Z., Y.S., and J.C.; Resources, W.C.; Software, J.T.; Supervision, G.L.; Validation, B.Y., C.Z. and J.D.; Writing – original draft, J.Z., W.C., and J.T.; Writing – review & editing, all authors. All authors have read and agreed to the published version of the manuscript. Acknowledgement The authors acknowledged the staff at Shanghai Jiaotong University School of Medicine affiliated Ruijin Hospital, Guangdong Provincial People’s Hospital and The Second Affiliated Hospital of Guangzhou Medical University. Data Availability All the data and material were true and available. The data is available from the corresponding author upon reasonable request. References Stevens, S. M. et al. Antithrombotic Therapy for VTE Disease. Chest 160 , e545–e608 (2021). Lee, L. H. DOACs – advances and limitations in real world. 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Clark, S. & Alcala‐Zermeno, J. L. Apixaban anti‐Xa levels in clinical practice: A case report. Br J Clin Pharmacol 90 , 2935–2938 (2024). Wu, H., Yu, Q., Jin, P., Huo, L. & An, J. Association of rivaroxaban plasma trough concentrations with clinical characteristics and outcomes. Front Pharmacol 16 , (2025). Hedeland, Y., Gustafsson, C. M., Touza, Z. & Ridefelt, P. Hemolysis interference in 10 coagulation assays on an instrument with viscosity‐based, chromogenic, and turbidimetric clot detection. Int J Lab Hematol 42 , 341–349 (2020). Ma, T., Tang, D. & Sun, W. A-148 Performance Evaluation ofthe Zybio EXT 4800: A Novel Optical-Magnetic Coagulation Analyzer. Clin Chem 70 , (2024). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6989437","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":489086869,"identity":"18ce665f-3fa4-4d41-8105-9219590b39b6","order_by":0,"name":"Juntao Zhao","email":"","orcid":"","institution":"Shanghai Jiaotong University School of Medicine affiliated Ruijin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Juntao","middleName":"","lastName":"Zhao","suffix":""},{"id":489086870,"identity":"3efe1abe-56ae-4e7c-baf8-c459297ec7cd","order_by":1,"name":"Wenhao Cui","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenhao","middleName":"","lastName":"Cui","suffix":""},{"id":489086871,"identity":"e2c2dbc1-0f5a-4c1b-a906-57ddb38a4609","order_by":2,"name":"Yuying Shi","email":"","orcid":"","institution":"The Second Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuying","middleName":"","lastName":"Shi","suffix":""},{"id":489086872,"identity":"04b1ecf3-838d-4619-845d-e2577fd3054c","order_by":3,"name":"Jingfeng Tong","email":"","orcid":"","institution":"Zybio.Inc","correspondingAuthor":false,"prefix":"","firstName":"Jingfeng","middleName":"","lastName":"Tong","suffix":""},{"id":489086873,"identity":"62cf3d36-e4ce-4f2d-9e30-4a81d8fc6e97","order_by":4,"name":"Yujing Yang","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yujing","middleName":"","lastName":"Yang","suffix":""},{"id":489086874,"identity":"43b4073f-1138-46d3-843d-045e09ff7f9e","order_by":5,"name":"Jingwen Chen","email":"","orcid":"","institution":"The Second Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingwen","middleName":"","lastName":"Chen","suffix":""},{"id":489086875,"identity":"4781ef20-69e5-4c46-ac05-69b3420457a6","order_by":6,"name":"Weiqi Chen","email":"","orcid":"","institution":"The Second Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weiqi","middleName":"","lastName":"Chen","suffix":""},{"id":489086876,"identity":"4ea986c3-1927-42a5-a916-d02af207c0b6","order_by":7,"name":"Ying Feng","email":"","orcid":"","institution":"The Second Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Feng","suffix":""},{"id":489086877,"identity":"d91bf116-0a83-43f2-a6cc-bfbf5997a18a","order_by":8,"name":"Caifang Zeng","email":"","orcid":"","institution":"The Second Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Caifang","middleName":"","lastName":"Zeng","suffix":""},{"id":489086878,"identity":"506ca7ad-f693-4219-93a8-7c5980c857c8","order_by":9,"name":"Jing Dai","email":"","orcid":"","institution":"Shanghai Jiaotong University School of Medicine affiliated Ruijin Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Dai","suffix":""},{"id":489086879,"identity":"f8a7ef4a-3c1b-4508-9826-452847d8eef0","order_by":10,"name":"Beixin Yu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Beixin","middleName":"","lastName":"Yu","suffix":""},{"id":489086880,"identity":"9527393c-8d4f-41cb-90bd-402aaa9315f0","order_by":11,"name":"Guanghua Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYBACPmYIncDAwHzgwMc/Njz8/A34tbAhtLAlPpzZkCYjOeMAAS0McC08xsa8DYdtDBoSCGhhZ78m8XNHbZ7B8QNmErw7zvMYMBxg/PAxB5/DeMoke88cLzY4k5AmIXnmNo85cwOz5MxteLWkSfC2HUvccCDhmIQB220ey4YDbMy8BLRI/gVpOf+wTSKB7RyPwYEEQlrYj0nzttUkbriRzGxwsO0AMVp4mK1l2w4kzrzxjPFhw5lkHskZB5vx+oWf//jDm2/b6hL7zud/OPynws6en7/54IePeLQAo8MASBxGFmFswKceCNgfAIk6AopGwSgYBaNgRAMAq8xWxlhDMewAAAAASUVORK5CYII=","orcid":"","institution":"Southern Medical University","correspondingAuthor":true,"prefix":"","firstName":"Guanghua","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-06-27 08:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6989437/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6989437/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87486148,"identity":"e53d699f-9e4e-4b7a-90d4-519b710e3033","added_by":"auto","created_at":"2025-07-24 11:00:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":72053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe flowchart of this study.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6989437/v1/901d8540c3ebc6019abe01ce.jpg"},{"id":87486149,"identity":"2a32c1fd-e8af-4017-ada9-e1637b88b121","added_by":"auto","created_at":"2025-07-24 11:00:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22265,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScatterplot of Riva anti-Xa compared to Riva LC-MS. \u003c/strong\u003eOverall Spearman’s correlation coefficient and regression line are shown; coefficients of the regression equation according to different strata of rivaroxaban plasma concentration are reported in Table 2.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6989437/v1/c17af4eba549a855079b6667.jpg"},{"id":87486150,"identity":"87019367-8a94-4ea4-b0d0-ab9e19315ebc","added_by":"auto","created_at":"2025-07-24 11:00:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":50579,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBland Altman plot for the differences between Riva LC-MS and Riva anti-Xa activity (units are ng/mL). \u003c/strong\u003eOverall bias and limits of agreement are shown; parameters according to different strata of rivaroxaban plasma concentration are reported in Table 2.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6989437/v1/ba875279bf1ef558db97d195.jpg"},{"id":87487137,"identity":"b5ac506f-80e5-4aec-96c1-68dd43c78637","added_by":"auto","created_at":"2025-07-24 11:08:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135442,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInterference assessment of the Riva anti-Xa assay with various potential interferents in plasma samples.\u003c/strong\u003e Each panel represents the effect of a specific interferent, namely bilirubin (A), hemoglobin (B), triglycerides (C), dabigatran (D), unfractionated heparin (UFH) (E), and low-molecular-weight heparin (LMWH) (F). The x-axis shows the concentration of the interferent, while the y-axis indicates the measured rivaroxaban concentration using Riva anti-Xa assay. Colored dots represent different spiked rivaroxaban concentrations (50 ng/mL in blue, 250 ng/mL in red, and 400 ng/mL in green), with each condition tested in triplicate. The percentage values denote the relative bias.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6989437/v1/102dee93f91e42a7c7ffff7e.jpg"},{"id":87486152,"identity":"bba8d327-436a-4920-a5b7-fca257bc6440","added_by":"auto","created_at":"2025-07-24 11:00:35","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":100033,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiagnostic accuracy of Riva anti-Xa assay for the measurement of rivaroxaban drug concentrations as determined in clinical practice.\u003c/strong\u003e (A) Box plots illustrating the distributions of measurements (median, interquartile range, minimum to maximum, all results) at clinically relevant cut-offs (30, 50, 100 ng/mL). (B) Receiver-operating characteristic curves (ROC) showing the diagnostic accuracy with sensitivity and specificity listed below.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6989437/v1/657d0822d922701bd02a7e0f.jpg"},{"id":95200640,"identity":"875b9eb0-78a4-4241-8097-0e5dafd5267e","added_by":"auto","created_at":"2025-11-05 12:09:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1431688,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6989437/v1/b8d4bd40-ada4-4f56-bf72-1c92d89357e1.pdf"},{"id":87487145,"identity":"305ae3ec-51e5-4131-a5c9-f2086189fbfb","added_by":"auto","created_at":"2025-07-24 11:08:36","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":398593,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6989437/v1/f8a49884e80311ee8df905f4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Multicenter Evaluation of a Rivaroxaban-Calibrated Anti- Xa Assay against LC-MS/MS in China: Correlation, Accuracy, and Clinical Utility","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRivaroxaban (Riva) is a direct oral anticoagulant (DOAC) that specifically inhibits both free and bound factor Xa activity, thereby blocking thrombin generation and preventing hemostasis. It has been approved for various clinical applications, including the prevention of venous thromboembolism (VTE) following hip or knee replacement surgery, the prevention of cardioembolic stroke in patients with non-valvular atrial fibrillation (NVAF), and the treatment of VTE, including pulmonary embolism (PE) and deep vein thrombosis (DVT) \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Numerous studies have demonstrated that DOACs, including rivaroxaban, outperform warfarin, and multiple professional associations recommend DOACs over warfarin for reducing stroke risk in patients with various indications \u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In terms of overall healthcare costs, rivaroxaban has also shown significant savings compared to warfarin\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough rivaroxaban exhibits clear and predictable pharmacological anticoagulant effects with fewer food and drug interactions compared to warfarin and does not require routine coagulation monitoring, understanding its plasma drug concentration is crucial in specific clinical scenarios. These scenarios include perioperative management, elderly patients, non-adherence and non-persistence, those with hepatic or renal impairment, planned thrombolysis, or major bleeding events \u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Additionally, real-world cohort studies have demonstrated substantial inter-individual variability in the plasma concentrations of rivaroxaban, influenced by factors such as renal function, age \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Furthermore, recent investigations have also highlighted potential interactions with commonly prescribed clinical medications, such as levetiracetam and valproic acid, which may affect rivaroxaban\u0026rsquo;s pharmacokinetics and anticoagulant efficacy\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRapid methods for detecting drug concentrations or factor Xa activity levels are essential for monitoring adherence, addressing potential treatment failure, ensuring the safety of perioperative anticoagulation management, and providing optimal care for long-term anticoagulated patients.\u003c/p\u003e\u003cp\u003eAlthough prothrombin time (PT) can serve as a surrogate marker for rivaroxaban efficacy, its correlation is suboptimal\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS, LC-MS) can accurately measure rivaroxaban concentrations within the range of 0.50 to 500 \u0026micro;g/L and is considered the gold standard for drug concentration assessment. However, its clinical utility is limited by slow turnaround times and restricted availability\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Anti-factor Xa (anti-Xa) activity assays address these limitations and present a viable option for clinical laboratories. Validated anti-Xa assays have been shown to reliably measure rivaroxaban concentrations\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe objectives of this study are: (1) to investigate the correlation between a commercial rivaroxaban anti-factor Xa assay (Riva anti-Xa, Zybio) and plasma drug concentrations measured by high-performance liquid chromatography-tandem mass spectrometry (Riva LC-MS); (2) to assess the interference resistance of the Riva anti-Xa assay by evaluating its performance in plasma samples spiked with potential interfering substances as well as clinical samples with or without interferents; and (3) to evaluate the diagnostic capability of the Riva anti-Xa assay in accurately predicting clinically relevant decision-making thresholds in practice.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eSpiking experiments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStandardized human plasma was spiked with varying concentrations of rivaroxaban reference standards. Rivaroxaban stock solutions were prepared in dimethyl sulfoxide (DMSO) and diluted to obtain eight final concentrations ranging from 20 to 520 ng/mL. The rivaroxaban anti-Xa activity assay was performed using a fully automated coagulation analyzer, EXT4800 (Zybio, Inc.).\u003c/p\u003e\u003cp\u003e\u003cb\u003eInterference resistance evaluation experiment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study evaluated the interference resistance of the Riva anti-Xa activity assay performed on the EXT4800 coagulation analyzer. The potential interfering substances included bilirubin, hemoglobin, triglycerides, dabigatran, unfractionated heparin (UFH), and low-molecular-weight heparin (LMWH). Each substance was added to standardized human plasma to achieve the following final concentrations: bilirubin at 0, 0.1, 0.2, and 0.3 mg/mL; hemoglobin at 0, 1.0, 1.5, and 2.0 mg/mL; triglycerides at 0, 5, 10, and 12 mg/mL; dabigatran at 0, 50, 250, and 500 ng/mL; UFH at 0, 1.0, and 2.0 IU/mL; and LMWH at 0, 2.0, and 4.0 IU/mL. For each level of interfering substance, low, medium, and high concentrations of rivaroxaban were added (final concentrations of 50, 250, and 400 ng/mL, respectively). Each prepared sample was measured three times using the Riva anti-Xa activity assay.\u003c/p\u003e\u003cp\u003eSamples without added interferents served as controls, and the relative bias of each interferent sample was calculated using the formula: Relative Bias (%) = (Mean of Interferent Group) - (Mean of Control Group)/(Mean of Control Group) \u0026times;100%.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStudy population\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis was a prospective multicenter clinical study that recruited patients on long-term rivaroxaban therapy in clinical practice between March, 2024 and September, 2024 in three Chinese tertiary hospitals. The flow chart of the patients is given in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All included patients provided written informed consent for study participation. Inclusion criteria were: age\u0026thinsp;\u0026ge;\u0026thinsp;18 years, current use of rivaroxaban (regardless of indication), and the ability to provide informed consent. Exclusion criteria included: pregnancy, age\u0026thinsp;\u0026lt;\u0026thinsp;18 years, incomplete sample information, and inability to provide informed consent. Data collection encompassed patient age, gender, indication for rivaroxaban, dosage and duration of rivaroxaban use, interference as well as other concomitant medications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSample size\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis clinical study was designed as a methodological comparison and used correlation coefficient testing (for continuous variables) to calculate the sample size. Following the CLSI EP9-A3 guidelines established by the Clinical and Laboratory Standards Institute (CLSI), the correlation coefficient between the test reagent results and the reference method should not be lower than 0.975. Based on the anticipated correlation coefficient of \u0026ge;\u0026thinsp;0.983 between the Riva anti-Xa assay and the Riva LC-MS results, the parameters were set as follows: α\u0026thinsp;=\u0026thinsp;0.025 (one-sided), s\u0026thinsp;=\u0026thinsp;1, β\u0026thinsp;=\u0026thinsp;0.2, P0\u0026thinsp;=\u0026thinsp;0.975, and P1\u0026thinsp;=\u0026thinsp;0.983. Using the sample size estimation formula for correlation coefficient testing (continuous variables) and calculated with PASS2021 software, the required sample size was determined to be 210 cases. Considering a 5% exclusion rate and the distribution of samples across three study centers, the final sample size for this clinical trial was set at 267 cases.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSample collection and processing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEach enrolled patient underwent a single blood draw. Using sterile techniques, one tube of blood was collected within 24 hours after drug administration in a 3.2% sodium citrate (blue-top) tube. Plasma was separated by standard clinical centrifugation methods, divided into two equal aliquots, and stored at -20\u0026deg;C. All samples were batch-tested at the end of the study.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnti-Xa activity assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA commercial Riva anti-Xa activity assay (Zybio) was employed using rivaroxaban calibrators and quality control materials. After rapid thawing, samples were gently mixed at 37\u0026deg;C. Patient plasma was pre-diluted (1:2, 100 \u0026micro;l) and mixed with a chromogenic substrate solution (250 \u0026micro;l). After incubation at 37\u0026deg;C, factor Xa (250 \u0026micro;l) was added. The reaction was terminated after 120 seconds, and absorbance was measured at 405 nm. All procedures were performed strictly according to the manufacturer's instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantification of rivaroxaban by LC-MS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRivaroxaban concentrations were quantified using LC-MS technology. To 50 \u0026micro;l of plasma, 10 \u0026micro;l of acetonitrile: water (1:1, v/v), 25 \u0026micro;l of extraction buffer, and 240 \u0026micro;l of precipitation reagent containing an internal standard were added to precipitate proteins and extract analytes. The mixture was centrifuged at 14,000 g for 4 minutes at 20\u0026deg;C. A 20 \u0026micro;l aliquot of the supernatant was diluted to 80 \u0026micro;l with water: methanol (8:2, v/v) and stored at 10\u0026deg;C until analysis. Calibrators and quality control samples were prepared using mixed plasma.\u003c/p\u003e\u003cp\u003eAnalysis was performed using reverse-phase chromatography on a triple quadrupole mass spectrometer (ABS4500 model). Rivaroxaban was separated by gradient elution at a flow rate of 0.4 ml/min, with the mobile phase consisting of 0.1% formic acid in water (A) and methanol (B).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe baseline characteristics and detection results of the enrolled patients were compiled into an Excel spreadsheet. Categorical variables were presented as case numbers and percentages, while continuous variables were summarized as medians with their 95% confidence intervals. Group comparisons were conducted using the Mann-Whitney U test.\u003c/p\u003e\u003cp\u003eThe relationship between Riva anti-Xa activity and Riva LC-MS was determined using Spearman correlation analysis. Bland-Altman plots were utilized to evaluate the agreement between Riva anti-Xa activity quantification and Riva LC-MS. Additionally, Deming regression was performed to assess the relationship between the two methods.\u003c/p\u003e\u003cp\u003eThe receiver operating characteristic curve (ROC) was plotted and the area under the curve (AUC), cut-off, sensitivity and specificity were calculated for clinically relevant drug concentrations of 30, 50, and 100 ng/mL \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAll statistical analyses and visualizations were conducted using GraphPad Prism version 10.1.2, with statistical significance set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEthics statement\u003c/b\u003e\u003c/p\u003e\u003cp\u003e The study protocol was approved by the Ethics Committees and institutional review boards of Shanghai Jiaotong University School of Medicine affiliated Ruijin Hospital (No: 2023\u0026thinsp;\u0026minus;\u0026thinsp;117), Guangdong Provincial People\u0026rsquo;s Hospital (No: QX2023-008-01) and The Second Affiliated Hospital of Guangzhou Medical University (No: Q2023-07-01). This study was conducted in accordance with the principles of the Declaration of Helsinki of the World Medical Association and with Good Clinical Practice Guidelines.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eSpiking experiments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA spiking experiment was conducted to evaluate the accuracy of the Riva anti-Xa activity assay. As shown in \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e, a strong linear relationship was observed between the actual concentrations of the spiked samples and those measured by the assay. The regression equation yielded a slope of 0.95 (95% CI: 0.86, 1.05), with a Spearman correlation coefficient of 1.0 between the two, indicating a high level of agreement.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBasic information of the enrolled participants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in the flowchart (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), we prospectively recruited patients on long-term rivaroxaban therapy who visited the three hospitals between March and September 2024. A total of 267 cases satisfying the inclusion and exclusion criteria were enrolled in this study with a median age of 61 years (95% CI: 59\u0026ndash;64), and 60.7% (162/267) were male. The demographic characteristics of the enrolled patients are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Among these patients, five were receiving concomitant heparin therapy at the time of sample collection. The majority of patients (47.2%, 126/267) were prescribed rivaroxaban for venous thromboembolism (VTE), while 28.1% (75/267) were being treated for atrial fibrillation (AF).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBasic information of the study population\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFeatures\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePatients (n\u0026thinsp;=\u0026thinsp;267)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMedian (95% CI)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e61 (59, 64)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGender (n, %)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e162 (60.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e105 (39.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIndication (n, %)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAtrial fibrillation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e75 (28.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVenous thromboembolism\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e126 (47.2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOthers\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e66 (24.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRivaroxaban dose (n, %)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.5 mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19 (7.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5 mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6 (2.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10 mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e124 (46.4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15 mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e98 (36.7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e20 mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20 (7.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eInterference (n, %)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e43 (16.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e224 (83.9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAccuracy of Rivaroxaban anti-Xa activity measurements\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOverall correlation between Riva anti-Xa activity results and rivaroxaban plasma concentration as determined by HPLC-MS (Riva LC-MS) was strong with a Spearmen\u0026rsquo;s correlation coefficient of 0.997 (95% CI, 0.996, 0.997) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, correlation was considerably lower at rivaroxaban plasma concentrations above 200 ng/mL (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The overall bias of the Bland-Altman difference plot is 0.405 with 95% agreement limit from \u0026minus;\u0026thinsp;16.2 to 17.0 ng/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Stratified analysis revealed a trend of increasing bias at higher rivaroxaban plasma concentrations (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAccuracy of Riva anti-Xa activity measurements if compared with Riva LC-MS\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRiva anti-Xa\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;50 ng/mL n\u0026thinsp;=\u0026thinsp;71\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50\u0026ndash;200 ng/mL n\u0026thinsp;=\u0026thinsp;134\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200 ng/mL n\u0026thinsp;=\u0026thinsp;62\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTotal n\u0026thinsp;=\u0026thinsp;267\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpearman\u0026rsquo;s correlation coefficient (95% CI)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.982 (0.970, 0.988)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.988 (0.983, 0.991)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.970 (0.951, 0.983)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.997 (0.996, 0.997)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003eDeming regression\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSlope (95% CI)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.997 (0.953, 1.040)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.03 (0.999, 1.06)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.03 (0.98, 1.08)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.003 (0.989, 1.018)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eY-intercept (95% CI)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.280 (-0.815, 1.376)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-2.28 (-5.30, 0.74)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-9.46 (-23.73, 4.82)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-0.015 (-1.379, 1.350)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003eBland-Altman difference plot\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBias (SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.178 (2.16)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.949 (6.88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-0.51 (14.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.41 (8.45)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLower limit of agreement\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-4.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-12.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-28.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-16.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUpper limit of agreement\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e17.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of Interference Resistance\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe ability to resist interference varies across different reagents and instrument platforms. The EXT4800 coagulation analyzer, as a hybrid system combining optical and magnetic detection technologies, offers both the sensitivity and precision of optical methods as well as the robust interference resistance of mechanical methods. In this study, we assessed the accuracy of the Riva anti-Xa assay in measuring rivaroxaban concentrations in interference-affected samples using the EXT4800 platform.\u003c/p\u003e\u003cp\u003eWe prepared samples containing varying concentrations of bilirubin, hemoglobin, triglycerides, dabigatran, unfractionated heparin (UFH), and low-molecular-weight heparin (LMWH), and spiked each with low, medium, and high concentrations of rivaroxaban (50 ng/mL, 250 ng/mL, and 400 ng/mL, respectively). Rivaroxaban concentrations were measured using the Riva anti-Xa activity assay, with samples without interferents serving as controls. The relative bias of measurements in the presence of each interferent was calculated accordingly.\u003c/p\u003e\u003cp\u003eAs shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the relative bias of rivaroxaban detection using the Riva anti-Xa activity assay remained within \u0026plusmn;\u0026thinsp;10% for bilirubin concentrations up to 0.3 mg/mL, with most biases within \u0026plusmn;\u0026thinsp;5% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). For hemoglobin concentrations up to 2.0 mg/mL, the relative bias remained within \u0026plusmn;\u0026thinsp;3% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Similarly, for triglyceride concentrations up to 12 mg/mL, the relative bias was within \u0026plusmn;\u0026thinsp;4% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt a dabigatran concentration of 500 ng/mL, the majority of the relative biases were within \u0026plusmn;\u0026thinsp;3.0%, except for a single measurement at the medium rivaroxaban concentration, which showed a bias of 9.4% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). For UFH concentrations up to 2.0 IU/mL, the relative bias was within \u0026plusmn;\u0026thinsp;2% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), and for LMWH concentrations up to 4.0 IU/mL, the relative bias was within \u0026plusmn;\u0026thinsp;3% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eWe further analyzed the batch of clinical samples, among which 43 samples were identified as having potential interferences, including hemolysis, jaundice, lipemia, or concomitant use of other anticoagulants. The differences between rivaroxaban concentrations measured by the Riva anti-Xa activity assay and Riva LC-MS in the interfering and non-interfering samples were compared. No significant differences were observed between the two groups (\u003cb\u003eFigure S2\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eThe correlation between the two detection methods remained high for both interfering and non-interfering samples, with correlation coefficients of 0.994 (95% CI: 0.989\u0026ndash;0.997) and 0.997 (95% CI: 0.996\u0026ndash;0.998), respectively (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results indicate that the Riva anti-Xa activity assay on the EXT4800 coagulation analyzer demonstrates strong resistance to interference when measuring rivaroxaban concentrations.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe influence of interference on the accuracy of Riva anti-Xa activity measurements if compared with Riva LC-MS.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWith interference, n\u0026thinsp;=\u0026thinsp;43\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWithout interference, n\u0026thinsp;=\u0026thinsp;224\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePearson\u0026rsquo;s correlation coefficient (95% CI)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.994 (0.989, 0.997)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.997 (0.996, 0.998)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eDeming regression\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSlope (95% CI)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.01 (0.985, 1.030)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.00 (0.987, 1.020)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eY-intercept (95% CI)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.011 (-2.55, 2.57)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.051 (-1.53, 1.43)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eBland-Altman difference plot\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBias (SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.944 (6.60)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.302 (8.77)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLower limit of agreement\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-12.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-16.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUpper limit of agreement\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDiagnostic performance of the Riva anti-Xa activity assay for predicting clinically relevant decision thresholds\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e illustrates the diagnostic accuracy of the Riva anti-Xa activity assay in identifying clinically relevant rivaroxaban concentrations. For a drug concentration threshold of \u0026ge;\u0026thinsp;30 ng/mL, the assay demonstrated a sensitivity of 100.0% (95% CI: 88.97\u0026ndash;100.0%) with an optimal cutoff value of 29.45 ng/mL, and a specificity of 99.58% (95% CI: 97.64\u0026ndash;99.98%). At a threshold of \u0026ge;\u0026thinsp;50 ng/mL, the sensitivity remained 100.0% (95% CI: 94.87\u0026ndash;100.0%) with a cutoff value of 49.70 ng/mL, and the specificity was 99.49% (95% CI: 97.17\u0026ndash;99.97%). For a threshold of \u0026ge;\u0026thinsp;100 ng/mL, the sensitivity was 92.74% (95% CI: 86.78\u0026ndash;96.13%) with a cutoff value of 95.70 ng/mL, and the specificity reached 99.30% (95% CI: 96.15\u0026ndash;99.96%). The area under the receiver operating characteristic (ROC) curve (AUC) for all three thresholds was 0.99, indicating excellent diagnostic performance of the Riva anti-Xa activity assay in distinguishing clinically relevant rivaroxaban concentrations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough rivaroxaban, a direct oral anticoagulant (DOAC), was introduced in China as early as 2009, a simple and practical method for measuring its plasma concentration in routine clinical laboratories has remained unavailable. While such assays are well-established internationally, their implementation in China has been limited. The Riva anti-Xa assay evaluated in this study aims to bridge this gap. To our knowledge, this is the first study to assess the accuracy, diagnostic performance, and interference resistance of this commercial assay in a Chinese clinical setting.\u003c/p\u003e\u003cp\u003eAs the number of patients using DOACs continues to rise, assessing medication adherence is becoming increasingly important. In other clinical scenarios, such as perioperative evaluations for major or critical organ surgeries, renal dysfunction, extreme body weight, and advanced age, therapeutic drug monitoring is essential to optimize anticoagulation therapy and minimize adverse events\u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. A case report from the Mayo Clinic described an elderly patient with an abnormally prolonged half-life of apixaban, emphasizing the potential impact of patient-specific factors on drug metabolism and clearance\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Similarly, Wu et al. analyzed a cohort of nearly 300 patients receiving rivaroxaban at Hebei Provincial People\u0026rsquo;s Hospital and reported an association between higher trough concentrations and an increased risk of bleeding events\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, further highlighting the potential value of continuous monitoring of anti-Xa levels in guiding anticoagulation management. Our study evaluated the accuracy and clinical diagnostic performance of the first Riva anti-Xa assay developed in China. The results demonstrated a high concordance with the reference method, underscoring its reliability and potential utility in routine clinical practice. These findings suggest that Riva anti-Xa assay could serve as a valuable tool for individualized anticoagulation management, particularly in patients with complex clinical conditions.\u003c/p\u003e\u003cp\u003eIn clinical practice, particularly before invasive procedures or surgeries, it is essential to discontinue anticoagulants. For patients undergoing procedures with a high risk of bleeding, determining the concentration of direct oral anticoagulants (DOACs) is a critical factor in guiding physician interventions. In patients requiring urgent high-risk interventions or experiencing life-threatening uncontrolled bleeding, a DOAC concentration of 50 ng/mL serves as the threshold for deciding whether to administer a DOAC reversal agent. Concentrations above this level necessitate the use of a reversal agent. Furthermore, in all scenarios, including elective surgeries, urgent interventions, or invasive procedures, DOAC concentrations must be below 30 ng/mL to minimize bleeding risk. For thrombolytic therapy consideration, a threshold of 100 ng/mL is critical. This study evaluated the diagnostic performance of the Riva anti-Xa activity assay at these three key clinical decision levels. Results demonstrated that the Riva anti-Xa activity assay exhibits high diagnostic performance in assessing concentrations at these clinically relevant thresholds, confirming its utility in clinical practice.\u003c/p\u003e\u003cp\u003eSample quality is critical for the analytical accuracy of laboratory diagnostics and patient safety. The most common pre-analytical interferences in coagulation laboratories involve hemolysis, jaundice, and lipemia in blood samples. While optical coagulation methods are highly sensitive and accurate, they are less robust against such interferences. Conversely, magnetic bead-based mechanical coagulation methods exhibit strong anti-interference capabilities but are less sensitive and accurate compared to optical methods. The interference resistance varies across different coagulation platforms. Hedeland et al. investigated the impact of hemolysis on 10 coagulation assays using the Stago STA R Max 2 analyzer and reported that hemolysis significantly interfered with anti-Xa activity measurement, with a hemoglobin concentration as low as 0.5 mg/mL leading to a 10% reduction in assay results \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In the present study, we employed the EXT4800 optical-magnetic integrated coagulation analyzer, which is designed to exhibit strong resistance to interference\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Our findings further confirmed its robustness, as even at a hemoglobin concentration of 2.0 mg/mL, the detection of Rivaroxaban anti-Xa activity remained unaffected in hemolyzed samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This demonstrates that the EXT4800 analyzer not only maintains reliable performance under challenging preanalytical conditions but also offers a potential advantage for routine coagulation testing in clinical laboratories.\u003c/p\u003e\u003cp\u003eThis study has several limitations that should be acknowledged. First, although the study included a robust sample size, it was limited to three centers, which may not fully represent the variability in sample handling and patient populations across different institutions. Second, while we assessed interference from hemolysis, jaundice, and lipemia, other potential pre-analytical and analytical factors, such as extreme pH or storage conditions, were not comprehensively examined. Additionally, our study did not include pediatric patients or those with rare coagulation disorders, which limits the generalizability of our findings to these populations. Finally, while the Riva anti-Xa assay demonstrated strong correlation and diagnostic performance against LC-MS, further studies are needed to evaluate its long-term clinical utility and cost-effectiveness in guiding anticoagulation management across diverse clinical scenarios.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study demonstrated that the Riva anti-Xa activity assay provides a highly accurate and reliable method for estimating rivaroxaban plasma concentrations, showing an excellent correlation with the gold-standard LC-MS method. The assay exhibited high sensitivity and specificity in identifying clinically relevant decision thresholds (\u0026ge;\u0026thinsp;30, 50, and 100 ng/mL), with ROC-AUC values of 0.99 across all thresholds. These findings support the Riva anti-Xa assay as a practical, efficient, and accessible tool for clinical laboratories, particularly in scenarios requiring rapid anticoagulation monitoring, such as perioperative management, high-risk bleeding events, and thrombolysis planning. Its simplicity and accuracy make it a valuable alternative to LC-MS for guiding rivaroxaban therapy in real-world clinical practice.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors have no competing interests to declare. The funding had no involvement in the study design, data collection, analysis, interpretation, or writing of the manuscript, and does not constitute a conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics declarations\u003c/h2\u003e\u003cp\u003eThe study protocol was approved by the Ethics Committees and institutional review boards of Shanghai Jiaotong University School of Medicine affiliated Ruijin Hospital (No: 2023\u0026thinsp;\u0026minus;\u0026thinsp;117), Guangdong Provincial People\u0026rsquo;s Hospital (No: QX2023-008-01) and The Second Affiliated Hospital of Guangzhou Medical University (No: Q2023-07-01).\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was funded by Guangzhou Science and Technology Project, grant number 2024A04J3705.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, G.L. and Y.F.; Data curation, J.D., J.Z., W.C.; Formal analysis, Y.S.; Funding acquisition, B.Y.; Investigation, Y.Y.; Methodology, J.T., J.Z., Y.S., and J.C.; Resources, W.C.; Software, J.T.; Supervision, G.L.; Validation, B.Y., C.Z. and J.D.; Writing \u0026ndash; original draft, J.Z., W.C., and J.T.; Writing \u0026ndash; review \u0026amp; editing, all authors. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors acknowledged the staff at Shanghai Jiaotong University School of Medicine affiliated Ruijin Hospital, Guangdong Provincial People\u0026rsquo;s Hospital and The Second Affiliated Hospital of Guangzhou Medical University.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll the data and material were true and available. The data is available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eStevens, S. M. \u003cem\u003eet al.\u003c/em\u003e Antithrombotic Therapy for VTE Disease. \u003cem\u003eChest\u003c/em\u003e \u003cstrong\u003e160\u003c/strong\u003e, e545\u0026ndash;e608 (2021).\u003c/li\u003e\n\u003cli\u003eLee, L. H. 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Hemolysis interference in 10 coagulation assays on an instrument with viscosity‐based, chromogenic, and turbidimetric clot detection. \u003cem\u003eInt J Lab Hematol\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 341\u0026ndash;349 (2020).\u003c/li\u003e\n\u003cli\u003eMa, T., Tang, D. \u0026amp; Sun, W. A-148 Performance Evaluation ofthe Zybio EXT 4800: A Novel Optical-Magnetic Coagulation Analyzer. \u003cem\u003eClin Chem\u003c/em\u003e \u003cstrong\u003e70\u003c/strong\u003e, (2024).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rivaroxaban, anti-Xa assay, LC-MS, correlation, interference resistance","lastPublishedDoi":"10.21203/rs.3.rs-6989437/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6989437/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackgrounds:\u003c/h2\u003e\u003cp\u003eRivaroxaban, a direct oral anticoagulant (DOAC) targeting Factor Xa, is widely used without routine monitoring. However, the absence of reliable drug level measurement may hinder clinical decision-making. The study evaluated the correlation between a commercial rivaroxaban-calibrated anti-Xa assay (Riva anti-Xa) and plasma drug levels measured by HPLC-MS/MS (Riva LC-MS), and assess its diagnostic accuracy for clinically relevant thresholds.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eA spiking experiment and a prospective multi-center study were conducted, using Riva LC-MS as the reference. Correlation and agreement were assessed via Spearman analysis, Bland-Altman plots, and Deming regression. Sensitivity and specificity were calculated of 30, 50, and 100 ng/mL.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe Riva anti-Xa assay showed excellent correlation with Riva LC-MS (Spearman r\u0026thinsp;=\u0026thinsp;0.982, 95% CI: 0.970\u0026ndash;0.988) and maintained acceptable bias in the presence of interfering substances. Clinical samples showed high concordance with LC-MS results. For concentrations\u0026thinsp;\u0026ge;\u0026thinsp;30, 50, and 100 ng/mL, the assay yielded sensitivities of 100.0%, 100.0%, and 92.74%, and specificities of 99.58%, 99.49%, and 99.30%, respectively. ROC-AUC values were 0.99 across all thresholds.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe Riva anti-Xa assay is a reliable, rapid alternative for estimating rivaroxaban levels, with strong correlation to LC-MS and excellent diagnostic accuracy, supporting its use in urgent clinical settings.\u003c/p\u003e","manuscriptTitle":"Multicenter Evaluation of a Rivaroxaban-Calibrated Anti- Xa Assay against LC-MS/MS in China: Correlation, Accuracy, and Clinical Utility","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-24 11:00:31","doi":"10.21203/rs.3.rs-6989437/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d687642a-a3e1-4568-a986-3fdb142dd508","owner":[],"postedDate":"July 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51922110,"name":"Health sciences/Biomarkers"},{"id":51922111,"name":"Health sciences/Diseases"},{"id":51922112,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-11-05T12:08:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-24 11:00:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6989437","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6989437","identity":"rs-6989437","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}