Thrombodynamics® analysis takes place in a single-use reaction cuvette upon initiation with an activation insert.  A key innovation of Thrombodynamics® is the use of time-lapse video-microscopy to analyze and quantify spatiotemporal parameters of fibrin growth and thrombin generation.

 

Thromobodynamics

 

Thrombodynamics® is both a qualitative and quantitative evaluation of blood plasma coagulation status by means of measuring and analyzing the spatiotemporal dynamics of clot growth and lysis 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 at a localized surface which has immobilized tissue factor mimicking a damaged blood vessel wall.

Unlike other routine coagulation assays the fibrin clot growth process in the 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, then propagates into the bulk of the plasma sample without interaction with the tissue factor bearing surface.

 

Competitive advantages of Thrombodynamics® test:

 

  • Ability to reveal plasma with either high thrombotic or a bleeding risk
  • Allows for study and analysis of the efficacy of anticoagulant drugs, including the newest generation of DOACs
  • Allows for study of efficacy of anti-platelet therapy
  • Ability to monitor and study thrombin generation
  • Allows for study and analysis of the efficacy of factor replacement drugs
  • An easy to understand and interpret global hemostasis assay
  • Based on the latest findings of biological science and nanotechnology
  • Small 120 µL sample volume
  • 3 simple pipetting steps from plasma, 4 to include thrombin generation
  • Global assay sensitive to both hypo and hypercoaguable blood
  • 2 channels for parallel testing
  • Bar-Coded reagents
  • Specific and sensitive reagent kits
  • Windows-based software
  • Open system for research applications
  • Define new tests in the system software
  • On-board thermostat for precise temperature control
  • 1x Thrombodynamics® T2T Analyzer with Power Cable
  • 1x Thrombodynamics® Laptop
  • 1x Thrombodynamics® Mouse
  • 1x USB (A-B) Interface Cable
  • 1x RJ-45 Ethernet Interface Cable
  • 1x HUB USB-LAN
  • 1x Thrombodynamics® User Manual
  • 1x Thrombodynamics® Quick-Start Guide
  • 1x Thrombodynamics® Service Kit
  • Thrombodynamics® TDX Kit (TDX-10)
  • Thrombodynamics® PLS Kit (PLS-10)
  • Thrombodynamics® Control Kit (C3)
  • Thrombodynamics® Measurement Cuvette (CT-5)
  • Technoclone tPA Purified Protein (TC41072)
  • Technoclone uPA Purified Protein (TC42000)
  • Technoclone Coagulation Control N (5020040)
  • Technoclone Coagulation Control A (5021055)
  • Technothrombin® Thrombin Generation Assay (TGA) Control Low (5006332)
  • Technothrombin® Thrombin Generation Assay (TGA) Control High (5006322)
  • Hart Biologicals GPIIb/IIIa Antagonist (HB-5537-FG)
  • ASPItest (08115826001)
  • Hart Biologicals Arachidonic Acid (HB-5505-FG)
  • Hart Biologicals Aspirin (HB-5514-FG)
  • Hart Biologicals ADP (HB-5501-FG)
  • Hart Biologicals Prostaglandin E1 (HG-5515-FG)
  • Hart Biologicals Collagen (HG-5503-FG)
  • Hart Biologicals Ristocetin (HG-5507-FG)
  • RISTOtest (08116644001)
  • Hart Biologicals TRAP-6 (HB-5512-FG)
  • TRAPtest (08116679001)

The Thrombodynamics® research analyzer records and measures spatiotemporal characteristics of the fibrin clot formation and thrombin generation processes in a thin layer of unstirred blood plasma.  The coagulation cascade is initiated as soon as the plasma comes into contact with the localized and immobilized tissue factor (TF) and the fibrin clot starts growing from the end face of the activation insert growing into the bulk of the plasma, mimicking damaged vasculature.  The process of fibrin clot formation is recorded by the Thrombodynamics® T2T Analyzer using an adjustable time time-lapse video microscopy by means of a dark-field light scattering method.  The obtained series of photos show both the thrombin generation and how the form, size, and density of the fibrin clot changes over time and in space.

On the basis of the recorded photos, the Thrombodynamics® Analytical Software calculates the numerical parameters of spatiotemporal dynamics of

 

Fibrin clot formation:

  • Tlag [min]: Lag Time
  • Tsp [min]: Spontaneous Clot Formation Time
  • V [µm/min]: Rate of Clot Growth
  • Vi [µm/min]: Initial Rate of Clot Growth
  • Vst [µm/min]: Stationary Rate of Clot Growth
  • CS [µm]: Clot Size
  • D [a.u.]: Clot Density

 

Thrombin Generation:

  • Ast [AU/L]: Stationary Amplitude of Thrombin Peak
  • Vt [µm/min]: Rate of Thrombin Peak Propagation
  • ETP_ATG [AU*min/L]: Thrombin Potential of Activator Thrombin Generation
  • Cmax_ATG [AU/L]: Maximum Concentration of Activator Thrombin Generation
  • Lag_ATG [min]: Lag Time of Activator Thrombin Generation
  • Tmax_ATG [min]: Time to Thrombin Peak in Activator Thrombin Generation

 

ParameterDefinitionDescriptionInfluencing Factor
Tlag [min]: Lag TimeThe time from the beginning of the measurement until the first significant levels of fibrin can be detected.The initiation phase of coagulation.  Analogous to the prothrombin (clotting) time.This parameter is sensitive to the initial stage of blood coagulation and reactions of the extrinsic pathway.
Tsp [min]: Spontaneous Clot Formation TimeThe time that spontaneous clots appear in the sample which has had no contact with the activator insert.  Defined as the time to spontaneous clots reaches 5% of the overall measurement region.Characterizes clotting that is independent of the activator surface.  0 under normal conditions.Spontaneous clotting is induced by circulating activators, active coagulation factors, and microparticles.
V [µm/min]: Rate of Clot GrowthThe average rate of clot growth.  The default range is during the interval of 15-25 minutes after Tlag but is automatically adjusted in the presence of spontaneous clots.Characterizes the propagation stage of blood coagulationThis parameter is sensitive to all coagulation cascade reactions, including the contact pathway and excluding the initiation reactions of the extrinsic pathway.
Vi [µm/min]: Initial Rate of Clot GrowthThe average rate of clot growth calculated on the interval 2-6 minutes following Tlag.Like Tlag, it describes the initial stages of clot growth but in terms of spatial elongation rather than local increase in thrombin concentration.This parameter is more sensitive to the reactions and components of the propagation stage than is Tlag, yet retains sensitivity to the initiation reactions.
Vst [µm/min]: Stationary Rate of Clot GrowthThe average rate of clot growth in the interval of 15-25 minutes after Tlag.  It is not calculated in the presence of spontaneous clots.Characterizes the propagation stage of blood coagulationThis parameter is sensitive to all coagulation cascade reactions, including the contact pathway and excluding the initiation reactions of the extrinsic pathway.
CS [µm]: Clot SizeThe clot size at the 30th minute (default value) of measurement.An integral parameter characterizing overall fibrin clot formation reflecting overall coagulation cascade performance.Sensitive to all major components and processes of blood coagulation.
D [a.u.]: Clot DensityAn optical parameter equal to the intensity of light scattering from a fibrin clot and is proportional to the density of the fibrin clot mesh.Characterizes firmness and structure of the formed clot but cannot replace a direct measurement of fibrinogen.Sensitive to fibrinogen and Factor XIII activity.
 
Ast [AU/L]: Stationary Amplitude of Thrombin PeakThe maximal activity of thrombin in the fibrin formation zone, which propagates away from the activator surface as a moving peak.This characterizes the propagation stage of blood coagulation.Ast is sensitive to all components and processes of blood coagulation.
Vt [µm/min]: Rate of Thrombin Peak PropagationThe average rate of thrombin generationThis parameter characterizes the propagation phase of blood coagulationVt is sensitive to changes in the intrinsic pathway of blood coagulation; factors VIII, IX, XI, V, X, and thrombin concentration.  It is also sensitive to phospholipid vesicle concentration.
ETP_ATG [AU*min/L]: Thrombin Potential of Activator Thrombin GenerationThe area under the curve of activator thrombin generation.Characterizes the initial stages of blood coagulation and is equivalent to the total thrombin generated a distance of 0.05-.02 mm from the activator .ETP_ATG is sensitive to changes in both the intrinsic and extrinsic pathways of blood coagulation
Cmax_ATG [AU/L]: Maximum Concentration of Activator Thrombin GenerationThe maximum concentration of thrombin generated at the near activator areaThis characterizes the initial stage of blood coagulationCmax_ATG is sensitive to changes in both the intrinsic and extrinsic pathways of blood coagulation
Lag_ATG [min]: Lag Time of Activator Thrombin GenerationCalculated as the time from initiation of experiment to when thrombin activity reaches 20 AU/LThis characterizes the initial stage of blood coagulationLag_ATG is sensitive to the extrinsic pathway of blood coagulation
Tmax_ATG [min]: Time to Thrombin Peak in Activator Thrombin GenerationCalculated as the time from initiation of the experiment to the peak in thrombin generation as the near-activator areaThis characterizes the initial stage of blood coagulationThis is sensitive to changes in both the intrinsic and extrinsic pathways of blood coagulation

 

In addition to those, a new feature of the Thrombodynamics® assay is the ability to visualize and quantify the fibrinolysis process in the presence of plasmin activators.  For this, several extra parameters can be calculated by the software:

  • LOT [min]: Lysis Onset Time
  • LP [%/min]: Lysis Progression
  • CLT [min]: Clot Lysis Time
  • LTE [min]: Lysis Time Estimation
  • LI [%]: Lysis Index

 

ParameterDefinitionDescription
LOT [min]: Lysis Onset TimeThe time at which the lysis is calculated to have initiated.Describes the initial stages of fibrinolysis
LP [%/min]: Lysis ProgressionThe linear rate of light scattering intensity decrease as a percentage of the initial value in the 5 minutes following LOTCharacterizes the maximal functional activity of plasmin.
CLT [min]: Clot Lysis TimeThe time span from the maximum Clot density (lmax) and when it reaches 5% of lmax.Description of overall lysis activity.
LTE [min]: Lysis Time EstimationThe time span between the lmax and the point when the clot density is projected to be 0.Like CLT, but extrapolated to total clot dissolution based on the calculated LP.
LI [%]The relationship of clot density at time moment LOT+5 to the lmaxCharacterizes the percent of clot density remaining 5 minutes after LOT

 

Dashkevich, N. M., Ovanesov, M. V., Baladina, A. N., Karamzin, S. S., Shestakov, P. I., Soshitova, N. P., . . . Ataullakhanov, F. I. (2012, November 21). Thrombin Activity Propagates in Space During Blood Coagulation as an Excitation Wave. Biophysical Journal, 103(10), 2233-2240. doi:10.1016/j.bpj.2012.10.011

Kuprash, A. D., Shibeko, A. M., Vijay, R., Nair, S. C., Srivastava, A., Ataullakhanov, F. I., . . . Balandina, A. N. (2018, December 18). Sensitivity and Robustness of Spatially Dependent Thrombin Generation and Fibrin Clot Propagation. Biophysical Journal, 115(12), 2461-2473. doi:10.1016/j.bpj.2018.11.009

Panteleev, M. A., Dashkevich, N. M., & Ataullakhanov, F. I. (2015, October). Hemostasis and thrombosis beyond biochemistry: roles of geometry, flow and diffusion. Thrombosis Research, 136(4), 699-711. doi: https://doi.org/10.1016/j.thromres.2015.07.025

Zhalyalov, A. S., Panteleev, M. A., Gracheva, M. A., Ataullakhanov, F. I., & Shibeko, A. M. (2017). Co-ordinated spatial propagation of blood plasma clotting and fibrinolytic fronts. PLoS One, 12(7), e0180668. doi:10.1371/journal.pone.0180668