Thrombodynamics Test Principle
Thrombodynamics is both a qualitative and quantitative evaluation of the blood plasma sample coagulation status by means of measuring and analyzing the spatiotemporal dynamics of fibrin clot growth in a heterogeneous in vitro system.
The thrombodynamics method aims to imitate in vitro physiological and pathophysiological processes that occur in vivo during hemostatic plug formation or thrombosis. The coagulation process in thrombodynamics starts from a localized surface which has immobilized tissue factor mimicking blood vessel wall damage.
Unlike other routine coagulation assays the fibrin clot growth process in thrombodynamics assay develops in space and time rather than only in time. The Fibrin clot starts to form, growing from the tissue factor bearing surface, but then propagates into the bulk of the plasma sample without interaction with the tissue factor bearing surface.
Thrombodynamics method is enabled by the Thrombodynamics Analyzer System T-2. Pre-prepared blood plasma samples are placed into the channels of the special measurement cuvette. Then a special activating insert is immersed into the cuvette. The end-faces of the activating insert are covered with the special coating that contains tissue factor (TF) – the main physiological activator of coagulation. The end face of the activating insert mimics the damaged surface of a blood vessel:
The fundamental concept of thrombodynamics assay is described in: Hemostasis and thrombosis beyond biochemistry: roles of geometry, flow and diffusion.
The obtained series of photos shows how the form, size, and density of fibrin clot changes over time. On the basis of the recorded photos the Thrombodynamics Analytical Software calculates the numerical parameters of spatiotemporal dynamics of fibrin clot formation (Thrombodynamics parameters).As soon as the blood plasma sample comes into a contact with TF the coagulation process initiates and fibrin clot starts growing from the end face of the activating insert into the bulk of the plasma sample. The process of fibrin clot formation is recorded by Thrombodynamics Analyser T-2 in a time-lapse video microscopy mode by means of dark-field light scattering method. The digital camera of the T-2 Analyser takes a series of photos of the light scattering from the cuvette.
Thrombodynamics-4D Test Principle
Thrombodynamics-4D assay is a new generation of thrombodynamics assay that is enabled only by the T2-T model of Thrombodynamics Analyser System. In addition to registering fibrin clot growth from the immobilized coagulation activator, Thrombodynamics-4D simultaneously allows registering spatiotemporal dynamics of thrombin, the main enzyme of the coagulation cascade. Registration of thrombin formation is based on fluorescent microscopy principle. Fluorogenic substrate for thrombin is added to plasma sample. The fluorogenic substrate is a molecule of 7-amino-4-methylcoumarin (AMC) bound to short amino acid sequence, which is required for recognition of substrate by thrombin. When bound to the substrate, AMC do not have an effect on plasma optical properties. As a result of substrate cleavage by thrombin, free AMC appears in plasma and is able to fluoresce. The rate of AMC formation in each point is proportional to local thrombin concentration. On the basis of the recorded photos of AMC fluorescence the Thrombodynamics Analytical Software calculates the numerical parameters of spatiotemporal dynamics of thrombin generation (thrombodynamics-4D parameters).