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Dielectric permittivity change detects the process of blood coagulation: Comparative study of dielectric coagulometry with rotational thromboelastometry
Thrombosis Research 145 (2016) 3–11
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Full Length Article
Dielectric permittivity change detects the process of blood coagulation: Comparative study of dielectric coagulometry with rotational thromboelastometry Yoichi Otaki a, Yusuke Ebana b,⁎, Shunji Yoshikawa a, Mitsuaki Isobe a a b
Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Tokyo, Japan Life Science and Bioethics Research Center, Tokyo Medical and Dental University, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 28 April 2016 Received in revised form 18 June 2016 Accepted 29 June 2016 Available online 30 June 2016 Keywords: Coagulation Dielectric blood coagulometry Anticoagulant therapy
a b s t r a c t Background: Intravascular thrombus formation causes various cardiovascular diseases. To monitor coagulation is important for screening native status, prevention from bleeding and maintaining it within its therapeutic range. The prothrombin time and the activated partial thromboplastin time are widely used for assessment and recognized as the conventional methods. Prothrombin time methods employ enhancement of coagulation with thromboplastin. Since the laboratory data depend on the production lot and/or the manufacturer, the accurate methods are required for evaluation. Rotational thromboelastometry (ROTEM) is a method based on detection of the change in resistance to rotational movement during blood clotting, while dielectric blood coagulometry (DBCM) is a novel method for assessment of clotting by measuring the change of electrical permittivity. These methods are thus based on the technology for observation of different physical phenomena. The aim of this study was to compare parameters such as the clotting time obtained by ROTEM and DBCM to evaluate their clinical usefulness. Methods and results: ROTEM and DBCM parameters were measured in 128 patients. The ROTEM clotting time showed a significant positive correlation with the DBCM coagulation time (R = 0.707, p b 0.001). Comparison of the DBCM coagulation time between patients with and without anticoagulant therapy (including novel oral anticoagulants) revealed a significant difference (43.8 ± 11.9 min in the anticoagulant group vs 29.4 ± 8.3 min in the control group, p b 0.001). Evaluation of coagulation was equivalent with DBCM and ROTEM. Conclusions: The present study suggested that DBCM, a novel method for measuring blood clotting, could provide the detail assessment for the status of anticoagulant therapy. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Intravascular thrombus formation is associated with various cardiovascular conditions, such as stroke, coronary artery disease, venous thromboembolism, atrial fibrillation, and prosthetic heart valves [1–5]. During the process of hemostasis after vascular injury, 1) aggregation of platelets occurs through binding to exposed subendothelial collagen, 2) release of tissue factor activates the coagulation cascades, and 3) activated thrombin promotes further platelet aggregation and catalyzes the conversion of fibrinogen into fibrin to reinforce the soft thrombus with a firm fibrin network that coats the complex of aggregated platelets and blood cells. While these hemostatic processes are crucial for maintaining the integrity of the high pressure circulatory system, Abbreviations: ROTEM, rotational thromboelastometry; DBCM, dielectric blood coagulometry. ⁎ Corresponding author. E-mail address: [email protected] (Y. Ebana).
http://dx.doi.org/10.1016/j.thromres.2016.06.030 0049-3848/© 2016 Elsevier Ltd. All rights reserved.
excessive generation of thrombin triggered by continuous endothelial stimulation or inflammation can initiate pathologic thrombosis with dysregulation of hemostasis and the development of a hypercoagulable state. Although anticoagulants and antiplatelet agents are prescribed for prevention of thrombosis, these medications sometimes cause bleeding complications such as intracranial hemorrhage or gastrointestinal bleeding [6,7]. To monitor coagulation, the prothrombin time (PT) and the activated partial thromboplastin time (aPTT) are often used in clinical practice [8], however, these methods are not necessarily useful for assessing native coagulation status. Rotational thromboelastography (TEG) and rotational thromboelastometry (ROTEM®, Tem International, Munich, Germany) have recently been proposed as methods for assessing the hemostatic status by estimating clot formation, clot dissolution kinetics, and clot strength from the response to a continuously applied rotational force. Both methods generate parameters based on the strength of thromboplastin in blood, and have mainly been adopted in the fields of surgery and anesthesiology
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Y. Otaki et al. / Thrombosis Research 145 (2016) 3–11
Fig. 1. DBCM data. (A) Example of a 3-dimensional dielectric spectrum obtained according to the standard protocol. The AC frequency (Hz), time (t), and change of dielectric permittivity (ε′/ε′t = 0) are shown on the x, y, and z axes. The color changes from blue at a lower permittivity to red at a higher permittivity. (B) Time course of the change in dielectric permittivity at 10 MHz. A subject without anticoagulant therapy is shown in the upper panel and one with anticoagulant therapy is shown in the lower panel. The vertical line indicates the peak time, showing the peak time is shifted to the right in the lower panel.
as point-of-care tests [9]. Previous reports have shown the usefulness of these methods for evaluating coagulation in patients with severe bleeding and acute or old venous thromboembolic events [9,10]. Hayashi et al. have proposed dielectric blood coagulometry (DBCM) as a new technique for estimating whole blood coagulability by measuring the change in the dielectric permittivity of a blood sample as it coagulates. Because alternating electric current around 10 MHz is very sensitive to the heterogeneity of the distribution and morphology of blood cells, the dielectric permittivity of whole blood gradually alters
with clot formation, reflecting changes of the cells assembled in the clot [11,12]. In a previous study, the coagulation time obtained by DBCM showed a good correlation with the clotting time (CT) obtained by a rheological method, thus confirming that the changes of dielectric permittivity corresponded to the progression of coagulation. This method enables us to evaluate the physiological process of blood coagulation from a new perspective. In the present study, we examined the correlation of DBCM findings with conventional laboratory tests (PT and aPTT), and also with ROTEM, as well as the clinical usefulness of DBCM for monitoring anticoagulant therapy.
Table 1 Clinical characteristics of study patients.
Male, n (%) Age (mean ± SD) Hypertension Dyslipidemia Diabetes mellitus Chronic kidney disease Current smoking Old myocardial infarction Atrial fibrillation Low EF (LVEF b 35%) Prior stroke Prior DVT Prior PCI/CABG Prior valvular op. History of malignancy Aspirin Clopidogrel Ticlopidine Warfarin Dabigatran Rivaroxaban
Control (n = 26)
Antiplatelet (n = 44)
Anticoagulation (n = 52)
Antiplatelet + anticoagulation (n = 6)
14 (54) 58.7 ± 20 14 11 5 1 1 0 3 1 0 1 0 0 1 0 0 0 0 0 0
22 (50) 62.9 ± 18 27 14 10 7 3 9 3 3 6 0 23 1 2 41 17 2 0 0 0
34 (65) 68 ± 12 28 22 12 7 6 0 44 2 6 3 0 7 8 0 0 0 45 5 2
5 (83) 71 ± 8 3 3 1 1 1 2 2 1 1 0 3 1 2 6 0 0 5 1 0
Abbreviations: LVEF = left ventricular ejection fraction, DVT = deep vein thrombosis, PCI = percutaneous coronary intervention, CABG = coronary artery bypass grafting.
Y. Otaki et al. / Thrombosis Research 145 (2016) 3–11
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Fig. 2. Comparison of DBCM coagulation time among four groups. (A) The patients were classified into four groups: (1) Control, (2) those prescribed antiplatelet drugs, (3) those using anticoagulants, and (4) those taking both classes of drugs. (B) Comparison of DBCM coagulation time between the control group and the anticoagulant group. (C) Evaluation of the coagulation time in patients using NOACs. (D) Evaluation of the clotting time in patients using warfarin and NOACs. There was significant difference in NATEM between control group and warfarin group. However, there was no statistical difference between control and NOACs group. Asterisks (* and **) indicate p b 0.001 and p b 0.05, respectively. NS; no significant difference.
2. Methods and materials
and bleeding events, which were defined as stroke, myocardial infarction, venous thromboembolism, intracranial hemorrhage, and gastrointestinal bleeding.
2.1. Study population This study was approved by the ethical committee of Tokyo Medical and Dental University (TMDU) in 2012. All subjects were recruited from the cardiovascular division of TMDU Hospital and gave written informed consent. The patients were fasting during blood collection, and not received unfractionated heparin or low-molecular-weight heparin administration. Patients were followed for at least six months after examination by DBCM to evaluate clinical events. Baseline and follow-up data were collected from medical records covering the period from 2012 to 2014. The primary outcome measures were thromboembolic
2.2. DBCM method In the present study, 128 individuals (including healthy controls) were tested by this method, and it was found that raw values were influenced by serum electrolytes and blood cells. Accordingly, the DBCM values were corrected for these factors. A venous blood sample was collected in a tube containing 0.2 ml of 3.13% sodium citrate as an anticoagulant. Within 5 h of collection, the blood was warmed for 10 min at 37 ° C, gently mixed by inversion, and set in a prototype dielectric
Table 2 Mean clotting time of DBCM and Other laboratory data.
Clotting time [min] NATEM [s] PT-INR aPTT [s] Platelet [×104/μl] Fibrinogen [mg/dl]
Control group
Anticoagulant group
p value
29.4 ± 8.3 723.0 ± 169.8 0.99 ± 0.08 29.9 ± 4.3 23.2 ± 7.0 322.7 ± 62.1
43.8 ± 11.9 961.3 ± 269.3 1.77 ± 0.48 40.2 ± 7.0 20.0 ± 5.1 318.0 ± 50.2
b0.001 b0.001 b0.001 b0.001 0.004 0.66
Abbreviations: PT-INR = prothrombin time-international normalized ratio, aPTT = activated partial thromboplastin time.
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Y. Otaki et al. / Thrombosis Research 145 (2016) 3–11
Fig. 3. Correlation of DBCM coagulation time with PT-INR and aPTT. Data for all 128 patients and subclassification based on medical treatment were plotted. The x-axis and the y-axis indicate the DBCM coagulation time and PT-INR or aPTT, respectively.
Y. Otaki et al. / Thrombosis Research 145 (2016) 3–11
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Fig. 3 (continued).
coagulation analyzer developed by Sony Corporation (Tokyo, Japan). The analyzer automatically dispensed 180 μl of blood into a disposable cartridge containing 12 μl of aqueous CaCl2 (200 mM), after which dielectric measurements were carried out at 37 °C over a frequency range from 100 Hz to 40 MHz for 60 min at 1-minute intervals. A gradual increase of permittivity (ε′/ε′t = 0) was observed as blood cells underwent aggregation. In a previous study, the peak time on the 10 MHz curve coincided with the initiation of blood clotting observed by rheological measurement. We defined the DBCM coagulation time as the endpoint of the major permittivity increase detected at 10 MHz which was sensitive to blood coagulation based on the previous report [12].
2.3. ROTEM method ROTEM was performed according to the manufacturer's standard procedure. Non-Activated TEM (NaTEM), In-TEM, Ex-TEM, and FibTEM (which evaluate the initial state, intrinsic coagulation system, extrinsic coagulation system, and fibrinolytic state, respectively) were employed for evaluation. The six main parameters determined by the ROTEM® method are the clotting time (CT, s), Clot Formation Time (CFT, s), alpha-angle, lysis index after 30 min (LI30), Maximum Lysis (ML), and Maximum Clot Firmness (MCF, mm). An increase in amplitude of 2 mm was defined as CT. Both CT and CFT are thought to indicate the coagulation time, while MCF and the alpha angle indicate clot
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firmness. Details of the DBCM and ROTEM methods have been reported elsewhere [9,11,12]. 2.4. Other parameters The platelet count and hematocrit were measured by automated methods (XN-20®, Sysmex, Kobe, Japan). Coagulation parameters such as the prothrombin time international normalized ratio (PT-INR), aPTT (s), and plasma fibrinogen level (mg/dl) were measured by a fully automated coagulation analyzer (CS-5100®, Sysmex, Kobe, Japan). 2.5. Statistical analysis Differences between groups were assessed by analysis of variance (ANOVA), followed by the Tukey-Kramer test. Continuous variables were presented as the mean ± standard deviation, while categorical variables were expressed as frequencies and were compared using the chi-square test or Fisher's exact test. Pearson's correlation analysis was employed to compare two parameters. Receiver operating characteristic (ROC) curves were calculated by considering patients with anticoagulants as true positive and patients without anticoagulant as true negative. ROC curves were expressed as area under the curves. Statistical analysis was performed with SPSS software version 21 (IBM SPSS Inc.), and p b 0.05 was considered to be statistically significant. 3. Results
displayed in Table 3. The follow-up rate was 96% and the median follow-up period was 182 days (interquartile range: 140–249 days). There were no thromboembolic or gastrointestinal bleeding events during the study period according to the ISTH classification [13]. 3.3. Comparison with ROTEM ROTEM is a commercially available method of evaluating blood clotting that is mainly used for patients with severe surgical bleeding, trauma, and other critical illnesses. It can be used to assess several factors related to the hemostatic status, such as platelet aggregation and coagulation, and there have been several reports of its efficacy. We compared the coagulation time obtained by DBCM with the clotting time obtained from several ROTEM parameters, including Non-Activated TEM (NaTEM), in-TEM, ex-TEM, and Fib-TEM. As a result, the DBCM coagulation time was correlated with the ROTEM clotting time shown by NaTEM (Pearson's coefficient r = 0.707, p b 0.001, Fig. 4) and Ex-TEM (r = 0409, p = 0.004, Fig. 4). However, there was no correlation of the DBCM coagulation time to In-TEM or fib-TEM (data not shown). This result indicates that DBCM could assess the initial state and the effect of the extrinsic coagulation system. In addition, the ROTEM clotting time shown by NaTEM demonstrated a significant difference between the control group and the warfarin group (p b 0.001) (Fig. 2D). Comparing DBCM with ROTEM by using ROC curve analysis regarding taking anti-coagulants, DBCM was superior to ROTEM in NaTEM assay (DBCM AUC 0.838, 95%CI: 0.768–0.908; p b 0.001 vs ROTEM AUC 0.779, 95%CI: 0.699–0.859; p b 0.001) (Fig. 5).
3.1. DBCM 4. Discussion A dielectric spectrum was obtained according to the manufacturer's instructions (Fig. 1A). We also investigated the difference of the coagulation time between untreated controls and subjects with anticoagulants (Fig. 1B). 3.2. Correlation between DBCM and current clinical methods A total of 128 patients recruited from TMDU hospital were examined by DBCM. Table 1 shows clinical characteristics of study patients at the time of enrollment. Their mean age was 64 ± 15 years and 36.5% were women. The patients were classified into the following four groups: (1) those using antiplatelet drugs, (2) those using anticoagulants, (3) those using both classes of drugs, and (4) those not using either. The mean coagulation time of the patients without antithrombotic agents was 30.39 ± 1.79 min. Mean coagulation time was prolonged in the second and third groups compared with the first and fourth groups (Fig. 2A). As shown in Table 2, DBCM revealed a difference of the mean coagulation time between patients using anticoagulants and controls (43.8 ± 11.9 and 29.4 ± 8.3, p b 0.001, Fig. 2B). We also evaluated the coagulation time in patients using novel oral anticoagulant agents. Fig. 2C shows a comparison of the coagulation time among patients using warfarin, those taking NOACs, and those without anticoagulants. It can be seen that the coagulation time of the patients taking NOACs is equivalent to that of those using warfarin and is prolonged in both groups relative to those without anticoagulants. As shown in Fig. 3, the results of standard tests such as PT-INR and aPTT were correlated with the CT value (r = 0.586 for PT-INR and 0.657 for aPTT, both p b 0.001). The patients prescribed anticoagulants (anticoagulant group, n = 58) were older (mean age: 68 ± 11 years) and more likely to have atrial fibrillation (73%), congestive heart failure (19.2%), and prior valvular surgery (9.6%), compared with the ones without anticoagulants. Prior percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) (31.1%) were significantly less frequent in the anticoagulant group. However, the prevalence of hypertension, diabetes mellitus, and chronic kidney disease did not show statistical differences between the two groups. Data on the 6-month clinical outcome are
Blood coagulation occurs through a complicated process in which various clotting factors and enzymes are involved. ROTEM and DBCM are promising methods for assessing the coagulation status based on curves that display the changes of different physical properties of whole blood. The main findings of the present study were as follows: 1) There was a positive correlation between the DBCM coagulation time and the ROTEM clotting time (NaTEM assay). 2) The DBCM coagulation time was also positively correlated with both PT-INR and aPTT. 3) The mean DBCM coagulation time was significantly longer in the anticoagulant group, including patients taking warfarin and NOACs, compared with the control group. First, we compared DBCM with the standard clinical assays. The DBCM coagulation time was moderately correlated with both PT-INR and aPTT, although the scatter plot showed that the correlation was not completely linear. To precisely assess clotting by measuring the PT or aPTT, correction or repeated examination is sometimes required and inconsistency occurs because these methods employ an aggregation inducer. Table 3 6-month clinical outcomes. Case no.
Group Clotting time [min] Platelet [×104/μl] PT-INR aPTT [s] NATEM [s] Days after examination Event
1
2
3
Control 24
Anticoagulant 32.8
Anticoagulant 37.6
35.2
17.9
26.1
0.98 26.4 710 209
1.3 36 723 313
2.43 47.2 915 222
Gastrointestinal Bleeding
Traumatic intracranial hemorrhage
Gastrointestinal Bleeding
Abbreviations: PT-INR = prothrombin time-international normalized ratio, aPTT = activated partial thromboplastin time.
Y. Otaki et al. / Thrombosis Research 145 (2016) 3–11
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Fig. 4. Correlation of the DBCM coagulation time with the ROTEM clotting time (NATEM and EXTEM assays). Data for all 128 patients and subclassification based on medical treatment were plotted below. Data on correlation of EXTEM with DBCM in combination group is not available since there is no data.
Comparison of ROTEM with DBCM revealed that the DBCM coagulation time was more closely correlated with ROTEM values (CT, CFT, and MCF) compared with PT data (Table 4) Therefore, it is considered that the DBCM coagulation time also reflects clot formation because of the correlation with ROTEM values. DBCM measures the permittivity of whole blood, which increased along with the process of coagulation after recalcification, and this is thought to reflect mainly the assembly of red and white blood cells after cross-linking with fibrin multimers. ROTEM directly assesses the time course of changes in clot elasticity as the fibrin network develops. Thus, these two methods assess the change
in the time-course of blood clotting from two different physical aspects of force and electricity, which showed that DBCM was more sensitive while on the other hand, ROTEM was more specific (Fig. 5). To investigate the clinical value of ROTEM and DBCM, we determined the mean coagulation time in patients taking warfarin or NOACs such as dabigatran and rivaroxaban, and we found a similar significant difference from the control group in both anticoagulant groups. Thus, the two new methods detected prolongation of the clotting time in patients using newer anticoagulant agents as well as those taking warfarin. In patients using dabigatran, aPTT was prolonged, while PT
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Y. Otaki et al. / Thrombosis Research 145 (2016) 3–11
Fig. 4 (continued).
was prolonged in patients using rivaroxaban. Therefore, these new methods may enable us to assess more accurately the overall coagulation status. Recently, several new methods have been developed for investigating the process of blood coagulation. Standard clinical methods such as PT and aPTT do not precisely reflect hypercoagulability and are unable to evaluate blood clotting comprehensively. While ROTEM assesses hemostasis and clot firmness from the viewpoint of viscoelastic changes,
DBCM could shed new light on the estimation of the risk of thrombus formation by detection of changes in electrical permittivity. Since the present study demonstrated that the DBCM coagulation time was positively correlated with both PT-INR and aPTT, DBCM may offer a standardized assessment system for coagulation. 5. Limitations This study had several limitations. First, the sample size was small. Second, selection of the subjects might have been affected by referralfilter bias. Thus, large-scale cohort studies are necessary for further evaluation of DBCM. Third, all the blood samples were collected during fasting at the TMDU hospital within 2 h after oral administration of drugs. However, we did not perform any special standardization for the collection of blood samples because the present study is an observational study. Table 4 Comparison of ROTEM with DBCM parameters.
DBCM vs NaTEM CT aPTT vs NaTEM CT PT-INR vs NaTEM CT DBCM vs NaTEM CFT aPTT vs NaTEM CFT PT-INR vs NaTEM CFT DBCM vs NaTEM MCF aPTT vs NaTEM MCF PT-INR vs NaTEM MCF
Fig. 5. ROC curve analysis of DBCM and ROTEM for assessing anticoagulant therapy.
r
p value
n
0.707 0.746 0.494 0.631 0.667 0.509 −0.403 −0.335 −0.27
b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001
128 124 125 128 124 125 128 124 125
Abbreviations: DBCM = dielectric blood coagulometry, NaTEM = non-activated thromboelastometry, CT = clotting time, aPTT = activated partial thromboplastin time, CFT = Clot Formation Time, PT-INR = prothrombin time-international normalized ratio, MCF = Maximum Clot Firmness.
Y. Otaki et al. / Thrombosis Research 145 (2016) 3–11
6. Conclusions The present study suggested that DBCM, a novel method for measuring blood clotting, could provide the detail assessment for the status of anticoagulant therapy. Acknowledgements The authors deeply thank Dr. Hayashi, Dr. Omori, and Mr. Brun for their advice on using DBCM equipment, which allowed us to obtain objective data. We also thank all of the participants in this study. Furthermore, we also thank the staff of the Departments of Cardiovascular Medicine and Life Science and Bioethics Research Center at TMDU. This research was supported by TMDU-Sony Research Support Fund (No. 2A169). References [1] R.G. Hart, J.L. Halperin, Atrial fibrillation and stroke: concepts and controversies, Stroke 32 (3) (2001) 803–808. [2] E.L. Pritchett, Management of atrial fibrillation, N. Engl. J. Med. 326 (19) (1992) 1264–1271. [3] R.A. Nishimura, C.M. Otto, R.O. Bonow, B.A. Carabello, J.P. Erwin 3rd, R.A. Guyton, P.T. O'Gara, C.E. Ruiz, N.J. Skubas, P. Sorajja, T.M. Sundt 3rd, J.D. Thomas, G. American College of Cardiology/American Heart Association Task Force on Practice, 2014 AHA/ACC guideline for the management of patients with valvular heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines, J. Am. Coll. Cardiol. 63 (22) (2014) e57–185.
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[4] P. Libby, P. Theroux, Pathophysiology of coronary artery disease, Circulation 111 (25) (2005) 3481–3488. [5] N.L. Browse, Prevention of venous thromboembolism, N. Engl. J. Med. 287 (3) (1972) 145–146. [6] R.G. Hart, O. Benavente, R. McBride, L.A. Pearce, Antithrombotic therapy to prevent stroke in patients with atrial fibrillation: a meta-analysis, Ann. Intern. Med. 131 (7) (1999) 492–501. [7] C. Antithrombotic Trialists, C. Baigent, L. Blackwell, R. Collins, J. Emberson, J. Godwin, R. Peto, J. Buring, C. Hennekens, P. Kearney, T. Meade, C. Patrono, M.C. Roncaglioni, A. Zanchetti, Aspirin in the primary and secondary prevention of vascular disease: collaborative meta-analysis of individual participant data from randomised trials, Lancet 373 (9678) (2009) 1849–1860. [8] W.H. Geerts, D. Bergqvist, G.F. Pineo, J.A. Heit, C.M. Samama, M.R. Lassen, C.W. Colwell, P. American College of Chest, Prevention of venous thromboembolism: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition), Chest 133 (6 Suppl) (2008) 381S–453S. [9] D. Whiting, J.A. Dinardo, TEG and ROTEM: technology and clinical applications, Am. J. Hematol. 89 (2) (2014) 228–232. [10] L. Spiezia, P. Marchioro, C. Radu, V. Rossetto, G. Tognin, C. Monica, L. Salmaso, P. Simioni, Whole blood coagulation assessment using rotation thrombelastogram thromboelastometry in patients with acute deep vein thrombosis, Blood Coagul. Fibrinolysis 19 (5) (2008) 355–360. [11] Y. Hayashi, Y. Katsumoto, S. Omori, A. Yasuda, K. Asami, M. Kaibara, I. Uchimura, Dielectric coagulometry: a new approach to estimate venous thrombosis risk, Anal. Chem. 82 (23) (2010) 9769–9774. [12] Y. Hayashi, M.A. Brun, K. Machida, M. Nagasawa, Principles of dielectric blood coagulometry as a comprehensive coagulation test, Anal. Chem. 87 (19) (2015) 10,072–10,079. [13] S. Schulman, C. Kearon, Subcommittee on Control of Anticoagulation of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis, Definition of major bleeding in clinical investigations of antihemostatic medicinal products in non-surgical patients, J. Thromb. Haemost. 3 (4) (2005) 692–694.
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Full Length Article
Dielectric permittivity change detects the process of blood coagulation: Comparative study of dielectric coagulometry with rotational thromboelastometry Yoichi Otaki a, Yusuke Ebana b,⁎, Shunji Yoshikawa a, Mitsuaki Isobe a a b
Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Tokyo, Japan Life Science and Bioethics Research Center, Tokyo Medical and Dental University, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 28 April 2016 Received in revised form 18 June 2016 Accepted 29 June 2016 Available online 30 June 2016 Keywords: Coagulation Dielectric blood coagulometry Anticoagulant therapy
a b s t r a c t Background: Intravascular thrombus formation causes various cardiovascular diseases. To monitor coagulation is important for screening native status, prevention from bleeding and maintaining it within its therapeutic range. The prothrombin time and the activated partial thromboplastin time are widely used for assessment and recognized as the conventional methods. Prothrombin time methods employ enhancement of coagulation with thromboplastin. Since the laboratory data depend on the production lot and/or the manufacturer, the accurate methods are required for evaluation. Rotational thromboelastometry (ROTEM) is a method based on detection of the change in resistance to rotational movement during blood clotting, while dielectric blood coagulometry (DBCM) is a novel method for assessment of clotting by measuring the change of electrical permittivity. These methods are thus based on the technology for observation of different physical phenomena. The aim of this study was to compare parameters such as the clotting time obtained by ROTEM and DBCM to evaluate their clinical usefulness. Methods and results: ROTEM and DBCM parameters were measured in 128 patients. The ROTEM clotting time showed a significant positive correlation with the DBCM coagulation time (R = 0.707, p b 0.001). Comparison of the DBCM coagulation time between patients with and without anticoagulant therapy (including novel oral anticoagulants) revealed a significant difference (43.8 ± 11.9 min in the anticoagulant group vs 29.4 ± 8.3 min in the control group, p b 0.001). Evaluation of coagulation was equivalent with DBCM and ROTEM. Conclusions: The present study suggested that DBCM, a novel method for measuring blood clotting, could provide the detail assessment for the status of anticoagulant therapy. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Intravascular thrombus formation is associated with various cardiovascular conditions, such as stroke, coronary artery disease, venous thromboembolism, atrial fibrillation, and prosthetic heart valves [1–5]. During the process of hemostasis after vascular injury, 1) aggregation of platelets occurs through binding to exposed subendothelial collagen, 2) release of tissue factor activates the coagulation cascades, and 3) activated thrombin promotes further platelet aggregation and catalyzes the conversion of fibrinogen into fibrin to reinforce the soft thrombus with a firm fibrin network that coats the complex of aggregated platelets and blood cells. While these hemostatic processes are crucial for maintaining the integrity of the high pressure circulatory system, Abbreviations: ROTEM, rotational thromboelastometry; DBCM, dielectric blood coagulometry. ⁎ Corresponding author. E-mail address: [email protected] (Y. Ebana).
http://dx.doi.org/10.1016/j.thromres.2016.06.030 0049-3848/© 2016 Elsevier Ltd. All rights reserved.
excessive generation of thrombin triggered by continuous endothelial stimulation or inflammation can initiate pathologic thrombosis with dysregulation of hemostasis and the development of a hypercoagulable state. Although anticoagulants and antiplatelet agents are prescribed for prevention of thrombosis, these medications sometimes cause bleeding complications such as intracranial hemorrhage or gastrointestinal bleeding [6,7]. To monitor coagulation, the prothrombin time (PT) and the activated partial thromboplastin time (aPTT) are often used in clinical practice [8], however, these methods are not necessarily useful for assessing native coagulation status. Rotational thromboelastography (TEG) and rotational thromboelastometry (ROTEM®, Tem International, Munich, Germany) have recently been proposed as methods for assessing the hemostatic status by estimating clot formation, clot dissolution kinetics, and clot strength from the response to a continuously applied rotational force. Both methods generate parameters based on the strength of thromboplastin in blood, and have mainly been adopted in the fields of surgery and anesthesiology
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Y. Otaki et al. / Thrombosis Research 145 (2016) 3–11
Fig. 1. DBCM data. (A) Example of a 3-dimensional dielectric spectrum obtained according to the standard protocol. The AC frequency (Hz), time (t), and change of dielectric permittivity (ε′/ε′t = 0) are shown on the x, y, and z axes. The color changes from blue at a lower permittivity to red at a higher permittivity. (B) Time course of the change in dielectric permittivity at 10 MHz. A subject without anticoagulant therapy is shown in the upper panel and one with anticoagulant therapy is shown in the lower panel. The vertical line indicates the peak time, showing the peak time is shifted to the right in the lower panel.
as point-of-care tests [9]. Previous reports have shown the usefulness of these methods for evaluating coagulation in patients with severe bleeding and acute or old venous thromboembolic events [9,10]. Hayashi et al. have proposed dielectric blood coagulometry (DBCM) as a new technique for estimating whole blood coagulability by measuring the change in the dielectric permittivity of a blood sample as it coagulates. Because alternating electric current around 10 MHz is very sensitive to the heterogeneity of the distribution and morphology of blood cells, the dielectric permittivity of whole blood gradually alters
with clot formation, reflecting changes of the cells assembled in the clot [11,12]. In a previous study, the coagulation time obtained by DBCM showed a good correlation with the clotting time (CT) obtained by a rheological method, thus confirming that the changes of dielectric permittivity corresponded to the progression of coagulation. This method enables us to evaluate the physiological process of blood coagulation from a new perspective. In the present study, we examined the correlation of DBCM findings with conventional laboratory tests (PT and aPTT), and also with ROTEM, as well as the clinical usefulness of DBCM for monitoring anticoagulant therapy.
Table 1 Clinical characteristics of study patients.
Male, n (%) Age (mean ± SD) Hypertension Dyslipidemia Diabetes mellitus Chronic kidney disease Current smoking Old myocardial infarction Atrial fibrillation Low EF (LVEF b 35%) Prior stroke Prior DVT Prior PCI/CABG Prior valvular op. History of malignancy Aspirin Clopidogrel Ticlopidine Warfarin Dabigatran Rivaroxaban
Control (n = 26)
Antiplatelet (n = 44)
Anticoagulation (n = 52)
Antiplatelet + anticoagulation (n = 6)
14 (54) 58.7 ± 20 14 11 5 1 1 0 3 1 0 1 0 0 1 0 0 0 0 0 0
22 (50) 62.9 ± 18 27 14 10 7 3 9 3 3 6 0 23 1 2 41 17 2 0 0 0
34 (65) 68 ± 12 28 22 12 7 6 0 44 2 6 3 0 7 8 0 0 0 45 5 2
5 (83) 71 ± 8 3 3 1 1 1 2 2 1 1 0 3 1 2 6 0 0 5 1 0
Abbreviations: LVEF = left ventricular ejection fraction, DVT = deep vein thrombosis, PCI = percutaneous coronary intervention, CABG = coronary artery bypass grafting.
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Fig. 2. Comparison of DBCM coagulation time among four groups. (A) The patients were classified into four groups: (1) Control, (2) those prescribed antiplatelet drugs, (3) those using anticoagulants, and (4) those taking both classes of drugs. (B) Comparison of DBCM coagulation time between the control group and the anticoagulant group. (C) Evaluation of the coagulation time in patients using NOACs. (D) Evaluation of the clotting time in patients using warfarin and NOACs. There was significant difference in NATEM between control group and warfarin group. However, there was no statistical difference between control and NOACs group. Asterisks (* and **) indicate p b 0.001 and p b 0.05, respectively. NS; no significant difference.
2. Methods and materials
and bleeding events, which were defined as stroke, myocardial infarction, venous thromboembolism, intracranial hemorrhage, and gastrointestinal bleeding.
2.1. Study population This study was approved by the ethical committee of Tokyo Medical and Dental University (TMDU) in 2012. All subjects were recruited from the cardiovascular division of TMDU Hospital and gave written informed consent. The patients were fasting during blood collection, and not received unfractionated heparin or low-molecular-weight heparin administration. Patients were followed for at least six months after examination by DBCM to evaluate clinical events. Baseline and follow-up data were collected from medical records covering the period from 2012 to 2014. The primary outcome measures were thromboembolic
2.2. DBCM method In the present study, 128 individuals (including healthy controls) were tested by this method, and it was found that raw values were influenced by serum electrolytes and blood cells. Accordingly, the DBCM values were corrected for these factors. A venous blood sample was collected in a tube containing 0.2 ml of 3.13% sodium citrate as an anticoagulant. Within 5 h of collection, the blood was warmed for 10 min at 37 ° C, gently mixed by inversion, and set in a prototype dielectric
Table 2 Mean clotting time of DBCM and Other laboratory data.
Clotting time [min] NATEM [s] PT-INR aPTT [s] Platelet [×104/μl] Fibrinogen [mg/dl]
Control group
Anticoagulant group
p value
29.4 ± 8.3 723.0 ± 169.8 0.99 ± 0.08 29.9 ± 4.3 23.2 ± 7.0 322.7 ± 62.1
43.8 ± 11.9 961.3 ± 269.3 1.77 ± 0.48 40.2 ± 7.0 20.0 ± 5.1 318.0 ± 50.2
b0.001 b0.001 b0.001 b0.001 0.004 0.66
Abbreviations: PT-INR = prothrombin time-international normalized ratio, aPTT = activated partial thromboplastin time.
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Fig. 3. Correlation of DBCM coagulation time with PT-INR and aPTT. Data for all 128 patients and subclassification based on medical treatment were plotted. The x-axis and the y-axis indicate the DBCM coagulation time and PT-INR or aPTT, respectively.
Y. Otaki et al. / Thrombosis Research 145 (2016) 3–11
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Fig. 3 (continued).
coagulation analyzer developed by Sony Corporation (Tokyo, Japan). The analyzer automatically dispensed 180 μl of blood into a disposable cartridge containing 12 μl of aqueous CaCl2 (200 mM), after which dielectric measurements were carried out at 37 °C over a frequency range from 100 Hz to 40 MHz for 60 min at 1-minute intervals. A gradual increase of permittivity (ε′/ε′t = 0) was observed as blood cells underwent aggregation. In a previous study, the peak time on the 10 MHz curve coincided with the initiation of blood clotting observed by rheological measurement. We defined the DBCM coagulation time as the endpoint of the major permittivity increase detected at 10 MHz which was sensitive to blood coagulation based on the previous report [12].
2.3. ROTEM method ROTEM was performed according to the manufacturer's standard procedure. Non-Activated TEM (NaTEM), In-TEM, Ex-TEM, and FibTEM (which evaluate the initial state, intrinsic coagulation system, extrinsic coagulation system, and fibrinolytic state, respectively) were employed for evaluation. The six main parameters determined by the ROTEM® method are the clotting time (CT, s), Clot Formation Time (CFT, s), alpha-angle, lysis index after 30 min (LI30), Maximum Lysis (ML), and Maximum Clot Firmness (MCF, mm). An increase in amplitude of 2 mm was defined as CT. Both CT and CFT are thought to indicate the coagulation time, while MCF and the alpha angle indicate clot
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firmness. Details of the DBCM and ROTEM methods have been reported elsewhere [9,11,12]. 2.4. Other parameters The platelet count and hematocrit were measured by automated methods (XN-20®, Sysmex, Kobe, Japan). Coagulation parameters such as the prothrombin time international normalized ratio (PT-INR), aPTT (s), and plasma fibrinogen level (mg/dl) were measured by a fully automated coagulation analyzer (CS-5100®, Sysmex, Kobe, Japan). 2.5. Statistical analysis Differences between groups were assessed by analysis of variance (ANOVA), followed by the Tukey-Kramer test. Continuous variables were presented as the mean ± standard deviation, while categorical variables were expressed as frequencies and were compared using the chi-square test or Fisher's exact test. Pearson's correlation analysis was employed to compare two parameters. Receiver operating characteristic (ROC) curves were calculated by considering patients with anticoagulants as true positive and patients without anticoagulant as true negative. ROC curves were expressed as area under the curves. Statistical analysis was performed with SPSS software version 21 (IBM SPSS Inc.), and p b 0.05 was considered to be statistically significant. 3. Results
displayed in Table 3. The follow-up rate was 96% and the median follow-up period was 182 days (interquartile range: 140–249 days). There were no thromboembolic or gastrointestinal bleeding events during the study period according to the ISTH classification [13]. 3.3. Comparison with ROTEM ROTEM is a commercially available method of evaluating blood clotting that is mainly used for patients with severe surgical bleeding, trauma, and other critical illnesses. It can be used to assess several factors related to the hemostatic status, such as platelet aggregation and coagulation, and there have been several reports of its efficacy. We compared the coagulation time obtained by DBCM with the clotting time obtained from several ROTEM parameters, including Non-Activated TEM (NaTEM), in-TEM, ex-TEM, and Fib-TEM. As a result, the DBCM coagulation time was correlated with the ROTEM clotting time shown by NaTEM (Pearson's coefficient r = 0.707, p b 0.001, Fig. 4) and Ex-TEM (r = 0409, p = 0.004, Fig. 4). However, there was no correlation of the DBCM coagulation time to In-TEM or fib-TEM (data not shown). This result indicates that DBCM could assess the initial state and the effect of the extrinsic coagulation system. In addition, the ROTEM clotting time shown by NaTEM demonstrated a significant difference between the control group and the warfarin group (p b 0.001) (Fig. 2D). Comparing DBCM with ROTEM by using ROC curve analysis regarding taking anti-coagulants, DBCM was superior to ROTEM in NaTEM assay (DBCM AUC 0.838, 95%CI: 0.768–0.908; p b 0.001 vs ROTEM AUC 0.779, 95%CI: 0.699–0.859; p b 0.001) (Fig. 5).
3.1. DBCM 4. Discussion A dielectric spectrum was obtained according to the manufacturer's instructions (Fig. 1A). We also investigated the difference of the coagulation time between untreated controls and subjects with anticoagulants (Fig. 1B). 3.2. Correlation between DBCM and current clinical methods A total of 128 patients recruited from TMDU hospital were examined by DBCM. Table 1 shows clinical characteristics of study patients at the time of enrollment. Their mean age was 64 ± 15 years and 36.5% were women. The patients were classified into the following four groups: (1) those using antiplatelet drugs, (2) those using anticoagulants, (3) those using both classes of drugs, and (4) those not using either. The mean coagulation time of the patients without antithrombotic agents was 30.39 ± 1.79 min. Mean coagulation time was prolonged in the second and third groups compared with the first and fourth groups (Fig. 2A). As shown in Table 2, DBCM revealed a difference of the mean coagulation time between patients using anticoagulants and controls (43.8 ± 11.9 and 29.4 ± 8.3, p b 0.001, Fig. 2B). We also evaluated the coagulation time in patients using novel oral anticoagulant agents. Fig. 2C shows a comparison of the coagulation time among patients using warfarin, those taking NOACs, and those without anticoagulants. It can be seen that the coagulation time of the patients taking NOACs is equivalent to that of those using warfarin and is prolonged in both groups relative to those without anticoagulants. As shown in Fig. 3, the results of standard tests such as PT-INR and aPTT were correlated with the CT value (r = 0.586 for PT-INR and 0.657 for aPTT, both p b 0.001). The patients prescribed anticoagulants (anticoagulant group, n = 58) were older (mean age: 68 ± 11 years) and more likely to have atrial fibrillation (73%), congestive heart failure (19.2%), and prior valvular surgery (9.6%), compared with the ones without anticoagulants. Prior percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) (31.1%) were significantly less frequent in the anticoagulant group. However, the prevalence of hypertension, diabetes mellitus, and chronic kidney disease did not show statistical differences between the two groups. Data on the 6-month clinical outcome are
Blood coagulation occurs through a complicated process in which various clotting factors and enzymes are involved. ROTEM and DBCM are promising methods for assessing the coagulation status based on curves that display the changes of different physical properties of whole blood. The main findings of the present study were as follows: 1) There was a positive correlation between the DBCM coagulation time and the ROTEM clotting time (NaTEM assay). 2) The DBCM coagulation time was also positively correlated with both PT-INR and aPTT. 3) The mean DBCM coagulation time was significantly longer in the anticoagulant group, including patients taking warfarin and NOACs, compared with the control group. First, we compared DBCM with the standard clinical assays. The DBCM coagulation time was moderately correlated with both PT-INR and aPTT, although the scatter plot showed that the correlation was not completely linear. To precisely assess clotting by measuring the PT or aPTT, correction or repeated examination is sometimes required and inconsistency occurs because these methods employ an aggregation inducer. Table 3 6-month clinical outcomes. Case no.
Group Clotting time [min] Platelet [×104/μl] PT-INR aPTT [s] NATEM [s] Days after examination Event
1
2
3
Control 24
Anticoagulant 32.8
Anticoagulant 37.6
35.2
17.9
26.1
0.98 26.4 710 209
1.3 36 723 313
2.43 47.2 915 222
Gastrointestinal Bleeding
Traumatic intracranial hemorrhage
Gastrointestinal Bleeding
Abbreviations: PT-INR = prothrombin time-international normalized ratio, aPTT = activated partial thromboplastin time.
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Fig. 4. Correlation of the DBCM coagulation time with the ROTEM clotting time (NATEM and EXTEM assays). Data for all 128 patients and subclassification based on medical treatment were plotted below. Data on correlation of EXTEM with DBCM in combination group is not available since there is no data.
Comparison of ROTEM with DBCM revealed that the DBCM coagulation time was more closely correlated with ROTEM values (CT, CFT, and MCF) compared with PT data (Table 4) Therefore, it is considered that the DBCM coagulation time also reflects clot formation because of the correlation with ROTEM values. DBCM measures the permittivity of whole blood, which increased along with the process of coagulation after recalcification, and this is thought to reflect mainly the assembly of red and white blood cells after cross-linking with fibrin multimers. ROTEM directly assesses the time course of changes in clot elasticity as the fibrin network develops. Thus, these two methods assess the change
in the time-course of blood clotting from two different physical aspects of force and electricity, which showed that DBCM was more sensitive while on the other hand, ROTEM was more specific (Fig. 5). To investigate the clinical value of ROTEM and DBCM, we determined the mean coagulation time in patients taking warfarin or NOACs such as dabigatran and rivaroxaban, and we found a similar significant difference from the control group in both anticoagulant groups. Thus, the two new methods detected prolongation of the clotting time in patients using newer anticoagulant agents as well as those taking warfarin. In patients using dabigatran, aPTT was prolonged, while PT
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Fig. 4 (continued).
was prolonged in patients using rivaroxaban. Therefore, these new methods may enable us to assess more accurately the overall coagulation status. Recently, several new methods have been developed for investigating the process of blood coagulation. Standard clinical methods such as PT and aPTT do not precisely reflect hypercoagulability and are unable to evaluate blood clotting comprehensively. While ROTEM assesses hemostasis and clot firmness from the viewpoint of viscoelastic changes,
DBCM could shed new light on the estimation of the risk of thrombus formation by detection of changes in electrical permittivity. Since the present study demonstrated that the DBCM coagulation time was positively correlated with both PT-INR and aPTT, DBCM may offer a standardized assessment system for coagulation. 5. Limitations This study had several limitations. First, the sample size was small. Second, selection of the subjects might have been affected by referralfilter bias. Thus, large-scale cohort studies are necessary for further evaluation of DBCM. Third, all the blood samples were collected during fasting at the TMDU hospital within 2 h after oral administration of drugs. However, we did not perform any special standardization for the collection of blood samples because the present study is an observational study. Table 4 Comparison of ROTEM with DBCM parameters.
DBCM vs NaTEM CT aPTT vs NaTEM CT PT-INR vs NaTEM CT DBCM vs NaTEM CFT aPTT vs NaTEM CFT PT-INR vs NaTEM CFT DBCM vs NaTEM MCF aPTT vs NaTEM MCF PT-INR vs NaTEM MCF
Fig. 5. ROC curve analysis of DBCM and ROTEM for assessing anticoagulant therapy.
r
p value
n
0.707 0.746 0.494 0.631 0.667 0.509 −0.403 −0.335 −0.27
b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001
128 124 125 128 124 125 128 124 125
Abbreviations: DBCM = dielectric blood coagulometry, NaTEM = non-activated thromboelastometry, CT = clotting time, aPTT = activated partial thromboplastin time, CFT = Clot Formation Time, PT-INR = prothrombin time-international normalized ratio, MCF = Maximum Clot Firmness.
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6. Conclusions The present study suggested that DBCM, a novel method for measuring blood clotting, could provide the detail assessment for the status of anticoagulant therapy. Acknowledgements The authors deeply thank Dr. Hayashi, Dr. Omori, and Mr. Brun for their advice on using DBCM equipment, which allowed us to obtain objective data. We also thank all of the participants in this study. Furthermore, we also thank the staff of the Departments of Cardiovascular Medicine and Life Science and Bioethics Research Center at TMDU. This research was supported by TMDU-Sony Research Support Fund (No. 2A169). References [1] R.G. Hart, J.L. Halperin, Atrial fibrillation and stroke: concepts and controversies, Stroke 32 (3) (2001) 803–808. [2] E.L. Pritchett, Management of atrial fibrillation, N. Engl. J. Med. 326 (19) (1992) 1264–1271. [3] R.A. Nishimura, C.M. Otto, R.O. Bonow, B.A. Carabello, J.P. Erwin 3rd, R.A. Guyton, P.T. O'Gara, C.E. Ruiz, N.J. Skubas, P. Sorajja, T.M. Sundt 3rd, J.D. Thomas, G. American College of Cardiology/American Heart Association Task Force on Practice, 2014 AHA/ACC guideline for the management of patients with valvular heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines, J. Am. Coll. Cardiol. 63 (22) (2014) e57–185.
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