Evaluation of the profile of thrombin generation during the process of whole blood clotting as assessed by thrombelastography

Abstract

The objective of this study was to evaluate the possibility of linking the tracing of whole blood clotting in a thrombelastograph (TEG) hemostasis system with the generation of thrombin assessed by thrombin/antithrombin complex (TAT). Citrated whole blood containing corn trypsin inhibitor from volunteers was clotted in the presence of CaCl2 and tissue factor. Clotting was monitored with the eight channels of a TEG system. At different time points, the whole blood TEG reaction cups were kept in a cold quenching solution, centrifuged, and the supernatants were kept at -80 degrees C until assayed for TAT by ELISA. The total thrombus generation (TTG) was calculated from the first derivative of the TEG waveform and was compared with thrombin generation measured by TAT. The two vector values--the TAT thrombin generation data and the corresponding TEG TTG--were analyzed using Pearson correlation coefficients (r) and linear, non-linear and natural log (ln) transformation of TAT values for least-squares goodness-of-fit curves. The best least-squares fit is an exponential curve. Linearizing using the ln of the TAT thrombin generation variable produces the same r (0.94) as of the exponential curve. The prediction equation is y = 8.0465 + 0.0005x (P < or = 0.0001), where y is the TAT thrombin generation variable in the ln transformation and x is the TEG TTG variable. The high magnitude of r and the high significance of the prediction equation demonstrate the high efficacy of the prediction of TAT thrombin generation by the use of TEG TTG.

Figure 1

The TEG^® analyzer measures the clot’s physical properties by the use of a special stationary cylindrical cup that holds a 360-μl sample of whole blood and is oscillated through an angle of 4º 45′. Each rotation cycle lasts ten seconds. A pin is suspended in the blood by a torsion wire and is monitored for motion. Thus, the magnitude of the output is directly related to the kinetics and the strength of the formed clot. As the clot retracts or lyses, these bonds are broken and the transfer of cup motion is diminished.

Figure 2

Schematic representation of TEG^® tracing with its principal parameters.

Figure 3

A representative TEG^® tracing obtained during the clotting process of whole blood of a normal subject. The shaded area represents the corresponding first derivative or velocity of the waveform produced by the TEG^® system. Several parameters corresponding to the rate of development of the tensile strength of the forming clot are derived from the first derivative of the waveform. The parameters are: MTG – Maximum Rate of Thrombus Generation (100*mm/sec) TTG – Total Area Under the Curve; Measures Total Thrombus Generation (mm) TMG – Time to Maximum Rate of Thrombus Generation (sec)

Figure 4

TEG^® segments representing sequential time points when the whole blood sample was stopped at various time intervals. The first derivative (velocity) of each segment determines the total thrombus generation, TTG, of that segment, while the corresponding TAT segment determines total TAT generation.

Figure 5

Linear regression through the origin of Total Thrombus Generation, the TTG variable, versus TAT Generation, the TAT variable.

Figure 6

Non-linear regression of Total Thrombus Generation, the TTG variable, versus TAT Generation, the TAT variable.

Figure 7

Linear regression of Total Thrombus Generation, the TTG variable, versus the natural log of TAT Generation, the TAT variable. The majority of the data-points are within the prediction intervals of the regression line.

Figure 8

Superposition of the TAT generation curve over the TEG^® tracing of whole blood from a normal control.