MODELLING THE ANTICOAGULATION PROCESSES AND DEVELOPING THE TECHNOLOGY FOR OPERATIVE ASSESSMENT OF FUNCTIONAL RESERVE OF HUMAN ANTICOAGULATION SYSTEM
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Аннотация
Introduction 7
1 Biological system of anticoagulation 10
1.1 Hemostasis and the anticoagulation factors 10
1.2 The role of endothelium in anticoagulation 16
1.3 Model of blood in study of anticoagulation 19
2 Methods for assessing the hemostasis 25
2.1 Laboratory methods 25
2.2 Thromboelastography in the assessment of hemostasis 28
2.3 Methods of vibration viscosimetry 32
3 Piezoelectric thromboelastography in the assessment of hemostasis 37
4 Studying the sensitivity of the piezotromboelastography technology 43
4.1 Physical fundamentals of vibrational viscosimetry 43
4.2 Study design 44
4.3 Study experiment 47
4.3.1 Calibration of the recording element 47
4.3.2 Amplitude-phase characteristics of the recording element 50
4.4 Analysis of the study results 56
5 Assessment technology of anticoagulation potential by LPTEG method 58
5.1 Determination of anticoagulation activity 58
5.2 New technology of the anticoagulation potential assessment 60
Conclusion 67
References 68
Appendix A. Patients data 73
Introduction 7
1 Biological system of anticoagulation 10
1.1 Hemostasis and the anticoagulation factors 10
1.2 The role of endothelium in anticoagulation 16
1.3 Model of blood in study of anticoagulation 19
2 Methods for assessing the hemostasis 25
2.1 Laboratory methods 25
2.2 Thromboelastography in the assessment of hemostasis 28
2.3 Methods of vibration viscosimetry 32
3 Piezoelectric thromboelastography in the assessment of hemostasis 37
4 Studying the sensitivity of the piezotromboelastography technology 43
4.1 Physical fundamentals of vibrational viscosimetry 43
4.2 Study design 44
4.3 Study experiment 47
4.3.1 Calibration of the recording element 47
4.3.2 Amplitude-phase characteristics of the recording element 50
4.4 Analysis of the study results 56
5 Assessment technology of anticoagulation potential by LPTEG method 58
5.1 Determination of anticoagulation activity 58
5.2 New technology of the anticoagulation potential assessment 60
Conclusion 67
References 68
Appendix A. Patients data 73
Developing the assessment technologies of functional reserves of human body is one of the most important tasks of the healthcare. Part of this task is the functional reserve assessment of the biological system regulating the aggregate blood state (RABS), and the links of this system, the main of which is hemostasis. The system RABS plays a key role in maintaining the homeostasis of the body, i.e. in selfregulation, in ability to support the constancy of its internal state through coordinated reactions aimed at maintaining dynamic equilibrium.
There are three main functional system of the complex system RABS: coagulation, anticoagulation, and fibrinolysis. Their interaction allows the hemostasis system to be kept within the limits of physiological fluctuations between hypo and hyper coagulation. Providing, on the one hand, the aggregative state of circulating blood, and on the other, preventing and stopping bleeding, the hemostasis system is one of the most labile systems of the body [1, 2].
The proper functioning of all components of the hemostasis is an indicator of its integrity. Operational and objective assessment of the functional state of the hemostasis system plays an extremely important role, since the late diagnosis of hemostatic disorders carries the potential threat of both thromboembolic and thrombohemorrhagic complications, often fatal (heart attacks, strokes, bleeding) [3]. Assessment of the anticoagulant potential (ACP), i.e. the functional reserve of the anticoagulant system (ACS), is an important task of clinical diagnosis.
Developing a technology for operational assessment of the ACS functional reserve is the aim of this work.
Currently, there are various diagnostic methods, techniques and technologies of the hemostasis laboratory testing in the clinical practice: amidolytic, clotting, enzyme immune assay. An important place is occupied by point-of-care testing (POCT) that are performed directly at the patient’s treatment site [4]. The goal of POCT is to collect the specimen and obtain accurate results in a very short period of time at the
location of the patient.
Most of the methods and technologies provide fragmentary information and do not allow to assess the state of the hemostasis as a whole system that functions comprehensively and inextricably within its links. [5]. The exceptions are global tests that characterize the result of the whole coagulation cascade. Global tests include thromboelastography (TEG), a method of graphically recording the processes of coagulation and fibrinolysis of whole blood [6, 7].
Whole blood has two main rheological properties, viscosity and plasticity [8], and belongs to the class of non-Newtonian fluids. Viscosity n(Y) is a non-linear function of the shear strain rate Y and depends on a number of factors: the concentration of blood cells and their aggregation parameters, the plasma composition and its spatial distribution, the rate of elastic shear deformations, external factors, and the mutual influence of various factors. Thus, blood is a multiphase and heterogeneous dispersed system, and belongs to nonlinear viscous- plastic media [9, 10].
Measuring the viscoelastic characteristics of whole blood is the basis of the TEG method. The further development of TEG became the methods of vibrational elastography, in particular the method of low-frequency piezotromboelastography (LPTEG). This technology is implemented in the ARP-01M Mednord analyzer. The analyzer measures the amplitude-temporal characteristics of the processes of coagulation, blood cells aggregation, and fibrinolysis. Thus, it is possible to assess the rheological properties of whole blood rapidly at all stages of hemocoagulation [6].
Currently, LPTEG is used to diagnose and monitor critical disorders of hemostasis [11], for instrumental assessment of the functional reserves of the hemostasis system [6, 12, 13]. The LPTEG method operates with amplitude-temporal characteristics of hemostasis processes, where the amplitude is measured in relative units (or the procedure defined units). The objective of this work is to evaluate the sensitivity of the LPTEG method in absolute values of the viscosity coefficient.
The study of blood by LPTEG is performed outside the influence of the organism, ex vivo, similar the study by classical thromboelastography. But unlike classical TEG, the method of low-frequency piezotromboelastography allows to investigate whole blood in vitro from the first seconds after taking it from vein with all the available internal reserve of pro- and anticoagulant substrates of blood. This allows to get the maximum number of parameters of the coagulation process, and, therefore, to evaluate the anti-coagulation potential more fully. Therefore, the use of LPTEG to assess the ACP is of particular practical importance.
Thus, in this research:
1) whole blood in-vitro is the main object of study;
2) the anticlotting mechanism and human anticoagulation system are the subject of research;
3) low-frequency piezoelectrical thromboelastograph, and oscilloscope, recording the amplitude and phase characteristics of the “sensor-blood” system, are the research tools.
Also the auxiliary objects are used in research: reference viscoelastic fluids, such as distilled water, and glycerol solutions of various concentrations (densities).
The research work is carried out on the basis of the laboratory "Modeling of physical processes in biology and medicine" of Physics Faculty of Tomsk State University.
The experimental stage is included the blood studies of patients, and it is conducted in Goldberg Research Institute of Pharmacology and Regenerative Medicine Clinic.
There are three main functional system of the complex system RABS: coagulation, anticoagulation, and fibrinolysis. Their interaction allows the hemostasis system to be kept within the limits of physiological fluctuations between hypo and hyper coagulation. Providing, on the one hand, the aggregative state of circulating blood, and on the other, preventing and stopping bleeding, the hemostasis system is one of the most labile systems of the body [1, 2].
The proper functioning of all components of the hemostasis is an indicator of its integrity. Operational and objective assessment of the functional state of the hemostasis system plays an extremely important role, since the late diagnosis of hemostatic disorders carries the potential threat of both thromboembolic and thrombohemorrhagic complications, often fatal (heart attacks, strokes, bleeding) [3]. Assessment of the anticoagulant potential (ACP), i.e. the functional reserve of the anticoagulant system (ACS), is an important task of clinical diagnosis.
Developing a technology for operational assessment of the ACS functional reserve is the aim of this work.
Currently, there are various diagnostic methods, techniques and technologies of the hemostasis laboratory testing in the clinical practice: amidolytic, clotting, enzyme immune assay. An important place is occupied by point-of-care testing (POCT) that are performed directly at the patient’s treatment site [4]. The goal of POCT is to collect the specimen and obtain accurate results in a very short period of time at the
location of the patient.
Most of the methods and technologies provide fragmentary information and do not allow to assess the state of the hemostasis as a whole system that functions comprehensively and inextricably within its links. [5]. The exceptions are global tests that characterize the result of the whole coagulation cascade. Global tests include thromboelastography (TEG), a method of graphically recording the processes of coagulation and fibrinolysis of whole blood [6, 7].
Whole blood has two main rheological properties, viscosity and plasticity [8], and belongs to the class of non-Newtonian fluids. Viscosity n(Y) is a non-linear function of the shear strain rate Y and depends on a number of factors: the concentration of blood cells and their aggregation parameters, the plasma composition and its spatial distribution, the rate of elastic shear deformations, external factors, and the mutual influence of various factors. Thus, blood is a multiphase and heterogeneous dispersed system, and belongs to nonlinear viscous- plastic media [9, 10].
Measuring the viscoelastic characteristics of whole blood is the basis of the TEG method. The further development of TEG became the methods of vibrational elastography, in particular the method of low-frequency piezotromboelastography (LPTEG). This technology is implemented in the ARP-01M Mednord analyzer. The analyzer measures the amplitude-temporal characteristics of the processes of coagulation, blood cells aggregation, and fibrinolysis. Thus, it is possible to assess the rheological properties of whole blood rapidly at all stages of hemocoagulation [6].
Currently, LPTEG is used to diagnose and monitor critical disorders of hemostasis [11], for instrumental assessment of the functional reserves of the hemostasis system [6, 12, 13]. The LPTEG method operates with amplitude-temporal characteristics of hemostasis processes, where the amplitude is measured in relative units (or the procedure defined units). The objective of this work is to evaluate the sensitivity of the LPTEG method in absolute values of the viscosity coefficient.
The study of blood by LPTEG is performed outside the influence of the organism, ex vivo, similar the study by classical thromboelastography. But unlike classical TEG, the method of low-frequency piezotromboelastography allows to investigate whole blood in vitro from the first seconds after taking it from vein with all the available internal reserve of pro- and anticoagulant substrates of blood. This allows to get the maximum number of parameters of the coagulation process, and, therefore, to evaluate the anti-coagulation potential more fully. Therefore, the use of LPTEG to assess the ACP is of particular practical importance.
Thus, in this research:
1) whole blood in-vitro is the main object of study;
2) the anticlotting mechanism and human anticoagulation system are the subject of research;
3) low-frequency piezoelectrical thromboelastograph, and oscilloscope, recording the amplitude and phase characteristics of the “sensor-blood” system, are the research tools.
Also the auxiliary objects are used in research: reference viscoelastic fluids, such as distilled water, and glycerol solutions of various concentrations (densities).
The research work is carried out on the basis of the laboratory "Modeling of physical processes in biology and medicine" of Physics Faculty of Tomsk State University.
The experimental stage is included the blood studies of patients, and it is conducted in Goldberg Research Institute of Pharmacology and Regenerative Medicine Clinic.
The research work has been accomplished with the achievement of the aim: the technology has been developed for the operative assessment of the functional reserve of the human anticoagulation system. It is demonstrated that the method of low- frequency piezoelectric thromboelastography is applicable for determining the anticoagulation potential of human blood under conditions of rapid diagnostics (point-of- care) in compliance with the basic requirements for hemostasis research methods (efficiency, informativity, reliability). It is shown that the maximum activity of the anticoagulant system is manifested in the initial stages of clot formation before the blood gelling point.
The objective of estimating the sensitivity of the LPTEG method is performed: it was confirmed that the method has the ability to determine the parameters of the piezothromboelastogram not only in relative units, but also in absolute values of the viscosity coefficient with appropriate calibration.
Laboratory experiments were carried out with different enzyme composition of blood under conditions of physiological norm and pathology (arterial hypertension of 1-2 degrees). The analysis of the obtained results confirmed the research hypothesis of the correlation of the viscoelastic properties of blood and its anti-coagulation activity.
The results of the research work confirm the possibility of using this approach to determine the viscoelastic properties of whole blood and to analyze their dynamics in the coagulation process in a way as close as possible to the study in vivo.
The operative assessment technology of the functional reserve of the human anti-coagulation system proposed in this work is registered as a patent application.
The objective of estimating the sensitivity of the LPTEG method is performed: it was confirmed that the method has the ability to determine the parameters of the piezothromboelastogram not only in relative units, but also in absolute values of the viscosity coefficient with appropriate calibration.
Laboratory experiments were carried out with different enzyme composition of blood under conditions of physiological norm and pathology (arterial hypertension of 1-2 degrees). The analysis of the obtained results confirmed the research hypothesis of the correlation of the viscoelastic properties of blood and its anti-coagulation activity.
The results of the research work confirm the possibility of using this approach to determine the viscoelastic properties of whole blood and to analyze their dynamics in the coagulation process in a way as close as possible to the study in vivo.
The operative assessment technology of the functional reserve of the human anti-coagulation system proposed in this work is registered as a patent application.
