Role of CES1 and ABCB1 Genetic Polymorphisms on Functional Response to Dabigatran in Patients with Atrial Fibrillation.

Background: Dabigatran etexilate is a pro-drug hydrolyzed into dabigatran by carboxylesterases (CES) and is a substrate of the P-Glycoprotein encoded by the adenosine-triphosphate-binding cassette sub-family B member (ABCB)1 genes. We evaluated the functional response to dabigatran according to different CES1 and ABCB1 single-nucleotide polymorphisms (SNPs) in patients with atrial fibrillation (AF). Methods: A total of 100 consecutive patients with AF taking dabigatran were enrolled by two Italian centers. A venous blood sample was drawn for genetic determinations, as well as a measurement of the diluted thrombin time (dTT) and drug plasma concentrations, at the trough and peak. The main objective was the relationship between the dTT values and CES1 rs2244613, CES1 rs8192935 and ABCB1 rs4148738 SNP while on two different dabigatran doses (110 and 150 mg BID). Results: A total of 43 patients were on a 110 mg dabigatran dose and 57 on 150 mg. The DTT values at the trough and at peak were not different among patients with different CES1 rs2244613 and CES1 rs8192935 genotypes, regardless of the dabigatran dose. In patients on 150 mg dabigatran, the dTT values at the trough were 77 (44-111) ng/mL in patients with the ABCB1 rs4148738 heterozygous CT genotype vs. 127 (85-147) ng/mL in the wild-type CC genotype vs. 110 (47-159) ng/mL in the mutant trait TT genotype (p = 0.048). In patients with the ABCB1 rs4148738 CT genotype, OR for having dTT values at a trough below the median was 3.21, 95% CI 1.04-9.88 (p = 0.042). Conclusions: ABCB1 rs4148738 CT heterozygous is associated with the reduced anticoagulant activity of dabigatran at the trough in patients receiving the higher dose regimen.

Comparisons between diluted thrombin time, ecarin chromogenic assays, and UPLC-MS for plasma level dabigatran quantification: Results from DRIVING study.

INTRODUCTION: The knowledge of dabigatran levels is helpful for decision-making in specific situations such as urgent surgery or when the question of reversal arises (uncontrolled bleeding, eligibility for thrombolysis). However, a limited number of observational studies are available regarding comparisons between quantification methods. The objective of the study was to compare dabigatran plasma levels using three assays including the reference method (high-performance liquid chromatography coupled with mass spectrometry), focusing on the agreement around the 30-50 ng/mL clinically relevant thresholds. METHODS: Sixty healthy volunteers from DRIVING trial (NCT01627665) were given a single 300-mg dabigatran etexilate dose. Serial blood samplings were performed at pre-defined time points (0 to 24 h). We analyzed plasma samples using ultra-performance-liquid chromatography coupled with tandem mass spectrometry (UPLC-MS) (dabigatran reference method); ii/diluted thrombin time (dTT) (Hemoclot-DTI-Hyphen-Biomed); iii/ecarin-based chromogenic assay (ECA-II-Stago). RESULTS: Nine hundred sixty samples were analyzed using the three assays (2759 values). dTT and ECA-II values were highly correlated with those of UPLC-MS (Deming regression). Most values >50 ng/mL were higher using dTT and ECA-II compared to UPLC-MS: biases were constant, +14% and +16% with dTT and ECA-II, respectively (Bland-Altman plots), suggesting that active metabolites accounted for ~15% of thrombin inhibition. Regarding values <30 ng/mL, 30-50 ng/mL, or ≥50 ng/mL, the agreement probability between dTT and ECA-II was of 90.6% [88.4-92.5] (Cohen's kappa coefficient 0.84). CONCLUSION: dTT and ECA-II assays rapidly provide accurate dabigatran-level results for clinical practice, both assays being suitable in emergency, taking into account the thrombin inhibitory effect of dabigatran metabolites.

International external quality assessment for measurements of direct oral anticoagulants: results and recommendations.

There is limited information regarding the performance of tests for direct oral anticoagulants (DOACs). To generate more knowledge, the accuracy of DOAC tests were evaluated using external quality assessment data from multiple years. This data demonstrated a good correlation for the tests with a small overall interlaboratory variability (10% for dabigatran, rivaroxaban and apixaban and 12% for edoxaban). The greatest differences between the various reagents were observed for rivaroxaban, especially for concentrations below 100 ng/ml. In conclusion, the results show overall reliable DOAC levels with some differences between reagent groups. Important finding: clinical decision criteria could be affected by the choice of reagent.

Dabigatran plasma levels, aPTT and thromboelastography in patients with AF: implications for allowing early non-elective surgical procedures.

According to current recommendations, patients on dabigatran should stop the drug 24-96 h before scheduled surgery. This may seem too long for non-elective cases. The aim of our study was to assess the number of patients on dabigatran who could theoretically undergo surgery 12 h post last drug dosing. We measured dabigatran plasma trough concentration by Hemoclot assay in 75 consecutive patients receiving dabigatran. Coagulation was assessed by aPTT and thromboelastography (TEG). Plasma levels ≤30 ng/ml were considered low. TEG parameters measured were clot reaction time (R), clot growth index (k), angle (α), maximal amplitude (MA) and the percentage of clot lysed after 30 min (LY30). Twelve patients (16%) had low plasma dabigatran levels 11.6 ± 0.9 h post last dosing. These patients compared to those with higher levels had significantly different aPTT (37.7 ± 4.4 vs. 49.6 ± 9.2 s; p < 0.001) and TEG R (6.7 ± 1.3 vs. 8.4 ± 2.6 min; p = 0.002). Only three of the patients with low levels had an aPTT >40 s. Within those with levels >30 ng/ml, four patients (6.4%) had plasma dabigatran levels ≥200 ng/ml, all with aPTT >65 s and TEG R >11 min. When the analysis was restricted to patients with creatinine clearance >80 ml/min, six (27.3%) had low plasma dabigatran levels. In this theoretical study, with a low risk population, it is suggested that one-sixth of patients receiving dabigatran have low drug concentrations at 12 h. Further studies are needed to confirm that such patients with low trough levels can actually undergo safely early surgery if necessary.

Performance verification of diluted thrombin time assay for detecting Dabigatran level in plasma / 临床检验杂志

Objective To evaluate the performance of diluted thrombin time (dTT) assay for detecting Dabigatran levels and observe whether this assay may meet the requirements of clinical laboratory.Methods According to EP15-A2,EP6-A,EP7-A and C-24 documents of the Clinical and Laboratory Standards Institute (CLSI),the precision,trueness,analytical measurement range,carryover rate and anti-biological interference of dTT assay were evaluated and the stability of specimen for dTT assay was observed.Results Both the within-day and between-day coefficient of variation (CV) of dTT assay for detecting Dabigatran levels were consistent with manufacturer's stated CV.Compared with target values of Dabigatran,the relative bias of 3 levels of proficiency test materials from College of American Pathologists (CAP) were less than 10％.The results meet linear verification when Dabigatran concentration was between 30.92 and 249.13 ng/mL.The carryover rate was-0.84％.There was no interference for Dabigatran levels by dTT assay for detecting Dabigatran when Hb≤3 g/L,triglyceride≤873 mg/dL,heparin≤2.2 IU/mL and FDP≤29 mg/L.The results of stability showed that plasma specimens for dTT could not be stored at room temperature more than 4 hours,at 4 ℃ more than 4 days,at-20 ℃ exceed 1 month,while at-80℃ the plasma specimens could be stored at least 6 months for dTT assay.Conclusion The precision,trueness,analytical measurement range,carryover rate,anti-biological interference of dTT assay may meet the requirement of clinical laboratory.The stability of the specimen can fulfill the clinical requirements.

Dabigatran en plasma: tiempos de trombina diluido, Ecarin y veneno de víbora de Russell diluido / Plasma dabigatran: diluted thrombin time, Ecarin and diluted Russell Viper Venom Time / Dabigatrana em plasma: tempos de trombina diluído, Ecarin e veneno de víbora de Russell diluído

Los objetivos del trabajo fueron verificar la calidad analítica del ensayo tiempo de trombina diluido (DTI) para medición de la concentración plasmática (cc) de dabigatran comparando dos coagulómetros de detección foto-óptica, comparar los resultados con el tiempo de Ecarin (ECT) y correlacionar las cc con las pruebas básicas de coagulación Tiempo de protrombina (TP), APTT y Tiempo de trombina (TT), y tiempo de veneno de víbora de Russell con fosfolípidos concentrados (DRVVTC). Se tomaron 43 muestras de plasma en el valle (10-14 h de la última toma) de 40 pacientes que recibían dabigatran. DTI y ECT presentaron (%) repetitividad <5,4% y <7,5%, CV interensayo <6% y <9%, respectivamente, en el protocolo EP15A2, aceptables para un Error Total permitido (TEa) <15%. Las cc medidas en pacientes fueron: mediana 83 (4-945) ng/mL. La comparación de equipos ACL TOP 300 y 500 dio resultados equivalentes por procedimiento alternativo de comparación de métodos. La comparación ECT vs. DTI fue satisfactoria por regresión de Deming (pendiente 1,143, ordenada al origen -19,33). Las correlaciones de cc vs. APTT, TP y DRVVTC fueron moderadas y no lineales tendiendo a plateau a cc>350 ng/mL, r2 0,59, 0,66 y 0,59, respectivamente. El TT fue extremadamente sensible: >120 s a cc 50 ng/mL. DTI presentó un buen desempeño analítico y permitió cuantificar dabigatran plasmático a cc bajas y altas en ambos equipos utilizados. ECT presentó resultados comparables a DTI. Se verifica una correlación moderada entre cc de dabigatran y las pruebas clásicas y DRVVTC, pudiendo ser estimadores de cc a partir de los 50 ng/mL.

The aims of the study were to verify the analytical performance of Dilute Thrombin Time (DTI) test to measure plasma dabigatran concentration (cc) in two photo-optical coagulometers, compare Ecarin clotting Time (ECT) and DTI results, and correlate cc with classical coagulation tests: prothrombin time (PT), APTT, thrombin time (TT) and diluted Russell Viper Venom Time tests with high phospholipid concentration (DRVVTC). Forty three plasma samples from 40 patients taking dabigatran were drown at through (10-14 hs.since last dose). DTI and ECT showed repetitivity (%) <5.4% and <7.5%, interassay CV <6% and <9%, respectively, following EP15A2 protocol, acceptable considering a Allowed Total Error (TEa)<15%. Patients` cc: median 83 (4-945) ng/mL. The comparison between ACL TOP 300 and 500 coagulometers showed equivalent results by using the alternative method comparison test. ECT vs. DTI: acceptable by Deming`s regression (slope 1.143, Y insert -19.33). cc vs. APTT, TP and DRVVTC: nonlinear and moderate correlations with plateau reached at cc >350 ng/mL, r2 0.59, 0.66 y 0.59, respectively. TT is extremely prolonged at cc >50 ng/mL. In conclusion: DTI showed a good analytical performance in both coagulometers. ECT showed comparable results to DTI. We verified that dabigatran cc presented moderate correlations with PT, APTT and DRVVTC, and that these tests could only qualitative estimate cc >50 ng/mL.

Os objetivos do trabalho foram verificar a qualidade analítica do ensaio tempo de trombina diluído (DTI) para medição da concentração plasmática (cc) de dabigatrana, comparando dois coagulômetros de detecção foto-óptica, comparar os resultados com o tempo de Ecarina (ECT) e correlacionar as cc com os testes básicos de coagulação Tempo de protrombina (TP), APTT e Tempo de trombina (TT), e tempo de veneno de víbora de Russell com fosfolipídios concentrados (DRVVTC). Foram tomadas 43 amostras de plasma no vale (10-14 h. da última toma) de 40 pacientes que recebiam dabigatrana. DTI e ECT apresentaram (%) repetitividade <5,4% e <7,5%, CV interensaio <6% e <9%, respectivamente, no protocolo EP15A2, aceitáveis para um Erro Total permitido (TEa) <15%. Cc medidas em pacientes: mediana 83 (4-945) ng/mL. Comparação de equipamentos ACL TOP 300 e 500: resultados equivalentes por procedimento alternativo de comparação de métodos. Comparação ECT vs. DTI: satisfatória por regressão de Deming (pendente 1,143, ordenada à origem -19,33). Correlações cc vs. APTT, TP e DRVVTC: moderadas e não lineares tendendo a plateau a cc>350 ng/mL, r2 0,59; 0,66 e 0,59, respectivamente. O TT é extremamente sensível: >120 s a cc 50 ng/mL. DTI apresentou um bom desempenho analítico e permitiu quantificar dabigatrana plasmática a cc baixas e altas em ambos os equipamentos utilizados. ECT apresentou resultados comparáveis com DTI. Verifica-se uma correlação moderada entre cc de dabigatrana e os testes clássicos e DRVVTC, podendo ser estimadores de cc a partir dos 50 ng/mL.

An update on laboratory measurements of Dabigatran: Smart specific and calibrated dedicated assays for measuring anti-IIa activity in plasma.

Use of Direct Oral Anticoagulants (DOACs) is continuously increasing for clinical application. The first product released was Dabigatran, which was proposed for many preventive and curative applications, especially for prevention of stroke in patients with non-valvular atrial fibrillation. Although measurement of Dabigatran Anti-IIa activity in plasma is not requested on a routine basis, in some situations its measurement is clinically useful. Especially, before an emergency surgery in treated patients, where its presence at high concentrations, which will expose the patient at an increased bleeding risk, has to be excluded. Hence, smart, specific, rapid and accurate quantitative assays are warranted as an essential required. Hemoclot™ Thrombin Inhibitors and Biophen® DTI were specifically designed for these applications, and can be used on all automated instruments with a standard range protocol for measuring concentrations at peak, or with a low range protocol for testing residual concentrations. Both functional assays have a good correlation with the reference LC-MS/MS method, and concentrations measured are similar. Performances of these assays and interferences of various substances or drugs are discussed. Some differences in variations of clotting times are observed between mechanical or optical clot detection instruments, which could be explained by the fibrin clot structure, altered by direct Factor Xa inhibitors, and more especially Rivaroxaban. Both clotting and chromogenic assays offer a safe and accurate quantitative measurement of Dabigatran in plasma in all situations where this determination is requested. In short this manuscript provides an in depth update on current opinions on laboratory aspects of measuring Dabigatran concentrations in plasma, when required.

Measurement of dabigatran plasma concentrations by calibrated thrombin clotting time in comparison to LC-MS/MS in human volunteers on dialysis.

INTRODUCTION: Dabigatran etexilate is an oral direct thrombin inhibitor. Although routine anticoagulation monitoring with dabigatran is not usually required, a simple and precise laboratory test to measure dabigatran concentrations in patient plasma may be useful in certain clinical circumstances, such as emergency situations. The HEMOCLOT(®) Thrombin Inhibitors assay has demonstrated accurate and precise determination of dabigatran concentrations within a range of 50-500 ng/ml. The objective of this study was to assess comparability of dabigatran concentrations determined by HEMOCLOT(®) and by liquid chromatography/tandem mass spectrometry (LC-MS/MS) in plasma samples from human volunteers with end-stage renal disease (ESRD) undergoing regular haemodialysis (HD) during a Phase I study. MATERIALS AND METHODS: Overall, 304 plasma samples were obtained from seven ESRD patients in dabigatran steady-state for measurement by HEMOCLOT(®) (calibrated diluted thrombin time [dTT]) and by LC-MS/MS. Agreement of dabigatran concentrations was assessed by regression analysis and difference plots. RESULTS: The measurements of calibration standards of the HEMOCLOT(®) assay showed excellent precision with coefficients of variation <5%. Accuracy determined by analysis of two quality control samples was 90% and 111%. HEMOCLOT(®)-derived dabigatran plasma concentrations paralleled those obtained by LC-MS/MS. The mean ratio of the LC-MS/MS and dTT-derived concentrations was 0.955 (67% limits of agreement: 0.771-1.18). CONCLUSIONS: The HEMOCLOT(®) Thrombin Inhibitors assay is suitable for measuring dabigatran plasma concentrations in volunteers with ESRD undergoing haemodialysis. The agreement between dabigatran concentrations determined by the HEMOCLOT(®) assay and the LC-MS/MS reference method met bioanalytical acceptance criteria.