Real-time visualization of clot growth
The main idea of Thrombodynamics assay is based on spatial separation of activation and propagation phases of coagulation.
The coagulation is activated by a special surface with immobilized tissue factor (TF), which reconstructs damage vessel wall. As soon as blood plasma sample comes into a contact with TF the coagulation process initiates and fibrin cot starts growing in a thin layer of non-stirred plasma, placed into a plastic cuvette.
Clot formation starts on the surface of the activator and propagates into the bulk of plasma. Images of clot formation are registered via CCD camera using a time laps microscopy mode in scattered light. The software then calculates the parameters of Thrombodynamics.
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.
|Tlag, [min]||Lag time||time between contact of plasma sample with activator and start of clot growth. Tlag characterizes the initiation phase of blood coagulation.|
|V, [µm/min]||Average rate of clot growth||The parameter characterizes the propagation phase of blood coagulation.|
|Tsp, [min]||Tsp is the time of spontaneous clots formation in a plasma sample volume, which had no initial contact with activating insert.||Spontaneous clotting is induced by circulating activators, active coagulation factors and microparticles.|
|Vi, [µm/min]||Initial rate||ICharacterizes the initiation phase of clot growth.|
|D, [a.u.]||Clot density,||Optical parameter, which is equal to intensity of light scattering from a fibrin clot.|
|CS, [µm]||Clot size on the 30th min of the measurement.|
Thrombodynamics Thrombin generation
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).
Thrombin generation and Fibrin generation
Ovaneson M.V. et al., Initiation and propagation of coagulation from tissue factor-bearing cell monolayers to plasma: initiator cells do not regulate spatial growth rate. Puschino, Russia