Thromboelastographic Methods to Detect Hypercoagulability.

Venous thromboembolic events (VTE) cause significant morbidity and mortality. Based on various studies, the incidence of VTE is greater than 275,000 new cases per year in the United States (Heit JA, J Thromb Haemost. 2005. 3:1611-17). A laboratory assay that predicts the development of VTEs in high-risk populations could have significant impact by potentially leading to prophylactic therapy and minimizing sequelae from such events. The thromboelastograph (TEG) is a point of care global hemostatic assay that utilizes whole blood to provide a continuous graphic record of the physical dynamics of a clot during fibrin formation and subsequent lysis. In general, the TEG has been used most extensively to predict bleeding in the settings of liver transplantation and cardiac bypass surgery. When employing the manufacturer's standard methods, the TEG is suited to demonstrate reduced clotting capability (“hypocoagulability”). Previous studies using the thromboelastograph to detect hypercoagulability have yielded inconsistent results as the methods and parameters studied have varied. We have developed a novel method whereby the baseline, i.e., normal, TEG curve and parameters, are “hypocoagulable,” i.e., similar to that of a hemophilia patient, making hypercoagulability easier to detect.

After assessing a variety of pre-analytical conditions including withholding activation with either kaolin, celite, or tissue factor, re-calcifying citrated samples with varying amounts of calcium chloride, and addition of varying amounts of corn trypsin inhibitor (CTI, an intrinsic pathway inhibitor), the following method performed best at achieving the desired hypocoagulable TEG curve and parameters. Whole blood was collected from healthy adult volunteers into 3.2% sodium citrate tubes that were pre-loaded with 300 micrograms of CTI. The blood was then placed into the TEG and re-calcified with calcium chloride (no activators were used), and the TEG was run until full clot formation. In order to demonstrate that this novel method is sensitive to increased thrombin generation (a surrogate marker for hypercoagulability), semi-logarithmic concentrations of recombinant human thrombin were added ex vivo to blood from healthy adult volunteers and the assays were again run until full clot formation. Data were collected and descriptive statistics calculated.

With the methods described above we were able to consistently create TEG curves that were hypocoagulable as compared to standard methods (see table below). The TEG curves of our novel method showed prolonged clot initiation (increased r time), slower clot propagation (increased k time and decreased angle), and fairly similar maximum clot strength (MA). The addition of increasing concentrations of thrombin showed increasing hypercoagulability as measured by TEG parameters (decreasing r time, decreasing k time, increasing angle and MA) and curves compared to baseline.

The results indicate that with manipulation of the pre-analytic conditions, normal individuals can have a profoundly “hypocoagulable” TEG curve and parameters making the assay sensitive to detecting hypercoagulability. In addition, the results demonstrate the sensitivity of this assay to increasing concentrations of thrombin, a surrogate marker of hypercoagulability. Future studies will apply these methods to populations at high risk for VTE and will aim to predict VTE in those patients.

No relevant conflicts of interest to declare.