Whole blood coagulation thrombelastographic profiles employing minimal tissue factor activation

Authors


J. Ingerslev, Center for Haemophilia and Thrombosis, Department of Clinical Immunology, Aarhus University Hospital, Skejby, Denmark, Brendstrupgaardsvej 100, DK-8200 Århus N, Denmark.
Tel.: +45 89495182; fax: +45 89495192; e-mail: j-ing@post3.tele.dk

Abstract

Summary.  We investigated whole blood coagulation by thrombelastography (TEG) employing activation with minute amounts of tissue factor (TF). Continuous raw data captured were transformed into novel parameters, such as the maximum velocity (MaxVel) and the time to maximum velocity (t,MaxVel) of whole blood clot formation. The courses of the whole blood clot development were very similar to thrombin generation curves reported in plasma. In this assay healthy women (n = 30) showed an earlier onset and an increased coagulation velocity compared to healthy men (n = 30). In patients with severe hemophilia, and persons undergoing thromboprophylaxis, distinctly abnormal coagulation profiles were observed with a decrease in the MaxVel, as well as a prolonged t,MaxVel. Changes appeared to be dependent on the nature and severity of the hemostatic deficit. Preliminary studies in patients substituted with recombinant factor VIIa demonstrated a marked change in the coagulation profile, in which the MaxVel and t,MaxVel shifted towards normal in a dose-dependent way. Data suggest that the whole blood coagulation TEG profile, following activation with minute amounts of TF, may reflect the hemostatic potential in patients suspected of impaired hemostasis.

In routine coagulation analysis, citrated plasma has predominantly been used for several decades. The initial formation of fibrin can easily be detected photometrically, and employing activated partial thromboplastin time (APTT) and prothrombin time (PT) methodologies, plasma has been widely utilized in diagnosis and monitoring of patients with major coagulation abnormalities. New understanding of the biochemistry of physiological hemostasis, in particular the recognition of the importance of tissue factor (TF)-dependent generation of thrombin has altered our view of the clotting process [1]. The importance of thrombin generation patterns in various coagulation abnormalities has been established [2,3]. Moreover, a significant role of the platelet in promoting thrombin generation has been widely appreciated [4,5].

While plasma contains a majority of the coagulation factors implicated in the coagulation process, whole blood includes phospholipid bearing cells and platelets with an important ability to support coagulation. Recent studies have pointed to the importance of the functions of platelets and leukocytes during coagulation and fibrinolysis [6,7]. Thus, choosing whole blood for coagulation analysis theoretically appears more favorable compared to study of plasma coagulation by PT- and APTT-based methods that include excessive activation. Furthermore, often un-physiological phospholipids are included in the assays to substitute for natural cell surface phospholipids.

In 1948, Hartert introduced the thrombelastographic principle (TEG) that record viscoelastical changes during coagulation [8]. Thrombelastographic analysis of coagulation can be carried out on plasma as well as on whole blood.

Initially, thrombelastography attracted most interest in research laboratories. Today, thrombelastography is utilized in several clinical settings, e.g. monitoring of hemostasis during liver transplantation and cardiac surgery [9]. Recently a newly developed portable TEG instrument (roTEG Coagulation Analyzer) has been marketed [10]. In this TEG, data are continuous, digital, and retrievable for further calculations.

Approaching the important role of platelets, cells, and TF in hemostasis, we have investigated a concept of overall patient-near hemostatic profiling. We hypothesized that the hemostatic capacity and its possible deficiencies might be disclosed and monitored by an activated whole blood method. We adopted a new system for data calculation and graphical display of whole blood clot formation profiles, whereby alterations in coagulation dynamics are accentuated and interpretation simplified. The aims of our study were to perform a preanalytic, an analytic, and a general biological assessment of the roTEG system utilizing methods based on activation with small amounts of TF.

Materials and methods

Equipment

Measurements were performed on two roTEG Coagulation Analyzers (Pentapharm®, Munich, Germany). All analyses were performed in roTEG all-plastic reaction cups as devised by the manufacturer. Polypropylene and polyethylene pipettes were used to handle reagents and blood.

Reagents and tissue factor solution

The whole blood samples in the reference series were activated by 20 µL solution of recombinant human TF dilution (Innovin®, Dade Behring, Marburg, Germany). For establishment of contact pathway reference intervals 20 µL of Synthasil (Haemoliance®, Rariton, NJ, USA) substituted TF as activator. In all biological evaluations we employed 20 µL TF that was prediluted 1 : 1000 using a sodium barbital buffer (sodium barbital 28.5 mmol L−1, pH = 7.4). The volume of reagents and whole blood in each experiment was 340 µL, thus the final TF dilution was 1 : 17 000 corresponding to a theoretical level of approximately 13 pg mL−1 (∼ 0.35 pmol L−1). Citrated whole blood was re-calcified by 20 µL 0.2 mol L−1 CaCl2.

PH and Ca2+ evaluation

Utilizing a Radiometer ABL-610 series (Radiometer, Copenhagen, Denmark), we measured soluble Ca2+ in plasma just after re-calcification as well as 120 min later. Furthermore, we continuously monitored pH for 120 min in a blood sample from one person.

Blood samples

Using minimum stasis and a 21-gauge butterfly needle, blood samples were drawn into citrated siliconized glass tubes (3 mL) mixing one part 3.8% trisodium citrate and nine parts of blood for subsequent coagulation analysis.

Blood from volunteers

Following informed consent, reference blood samples were drawn from healthy volunteers consisting of laboratory staff persons and blood donors. Each of the 60 volunteers participating in the study had a smooth cubital venipuncture performed. This reference group had a gender ratio of one, the average age of female was 39.4 years (SD 11.7) and the average age of males was 40.1 years (SD 12.8). For further biological evaluation whole blood samples were included from patients with hemophilia A and coagulopathy owing to treatment with vitamin-K antagonists and heparin. In some experiments patients received treatment with recombinant factor VIIa (rFVIIa; NovoSeven®, Novo Nordisk A/S, Denmark) to control bleeding. In others, rFVIIa had been added to samples ex vivo. In each case, the level of substitution is specified in respective legends.

roTEG thrombelastographic analysis

roTEG thrombelastography was performed on a coagulation analyzer controlled by a portable computer. A plastic pin was fixed to a rotating axis that was brought in contact with the mixture of the whole blood sample and reagents that were dispensed onto a plastic cup mounted on an aluminum cup holder, thermostatically controlled at 37 °C. The pin initially rotates at an angle, ω, of 5° alternating left and right during a fixed time period [10]. During coagulation resistance against movements of the pin was detected by a photo-sensor and registered continuously. Traditionally, the development of elasticity during clot formation is given by a metric amplitude (mm) proportional to ω, that was depicted on the computer screen (Fig. 1a). The clotting time (CT) was defined by the manufacturer as the time in seconds from Ca2+ activation of coagulation and until an increase in elasticity corresponding to 2 graphical mm was obtained on the ordinate. The clot formation time (CFT) has been defined by the manufacturer as the time in seconds passing while the elasticity increases from 2 mm to 20 mm on the ordinate. The maximum clot formation (MCF) expresses the maximum strength in millimeters of the final clot. The MCF has been shown to correlate with the platelet count and platelet function as well as with the concentration of fibrinogen [11,12]. During our initial work with the roTEG elasticity signal, we developed a different set of logistics for characterization of the dynamic profile of whole blood coagulation. The digital data representing the continuous registration of clot formation were extracted from the roTEG system and the entire time-course was differentiated. We here report on the assignment of three new parameters in the assessment of coagulation dynamic properties. The maximum velocity (MaxVel) of clot formation, the time to maximum velocity (t,MaxVel) of clot formation, and the area under the velocity curve (AUC). Figure 1(b,c) depict a roTEG signal profile based on the same course of the coagulation process as in Fig. 1(a) but here illustrated in the DyCoDerivAu™ software (AvordusoL, Risskov, Denmark). Figure 1(c) illustrates the first derivative (velocity profile). The MaxVel is the maximum rate of the clot formation and is marked with a horizontal arrow. The t,MaxVel is the time at MaxVel that is marked with a perpendicular arrow. AUC is the area under the velocity curve in Fig. 1(c), and equals the endpoint minus the starting point of the elasticity curve. Dependent upon the length of the measurement the AUC may express an equivalent to the MCF.

Figure 1.

Principal characteristics of thrombelastographic software parameters and first derivative parameters of whole blood clot formation. (a) Standard thrombelastographic tracking. CT is the clotting time, CFT is the clot formation time, and the MCF is the maximum amplitude of clot formation. (b) roTEG tracking imported into the DyCoDerivAu™ software. (c) Velocity profile, the first derivative of the roTEG thrombelastographic course. Maximum velocity (MaxVel, marked with a horizontal arrow) is the maximum rate of whole blood clot formation. Time to maximum velocity (t,MaxVel, marked with a perpendicular arrow) is the time until maximum velocity occurs. The area under the velocity curve (AUC, marked with gray color) indicates the maximum clot formation, an indirect measurement of clot strength.

Analytical set-up

Whole blood samples were processed as follows: (i) the freshly obtained sample rested 30 min at room temperature; (ii) 300 µL of well-mixed citrated blood was added to a prewarmed roTEG plastic cup followed by 2 min rest at 37 °C; (iii) 20 µL activator (TF or synthasil) solution was added followed by additional 2 min of incubation at 37 °C; (iv) roTEG recording was begun immediately after addition of 20 µL 0.2 mol L−1 of CaCl2 solution.

Data calculation and graphics

In all instances, we recorded the CT, CFT, and MCF as devised by the manufacturer. Additionally, using the DyCoDerivAu™ software, calculations of the MaxVel, the t,MaxVel, and the AUC were executed. Raw data were differentiated with respect to time to obtain the 1st derivative of the elasticity signal. Since the roTEG expresses the elasticity by the unit of millimeter (mm), the first time derivative corresponds to a velocity signal. Knowing the time resolution, i.e. the Δt (s) between each recording point it is possible to determine the maximum velocity and the time of its occurrence. In all data analyses, the derived signals were low pass filtered to remove glitches and small spikes originating from minor noise. The velocity signal was integrated to give the AUC.

Statistical analysis

Mean values of the standard roTEG parameters and the DyCoDerivAu™ Software parameters were tested by an unpaired t-test comparing females and males. Significant level was set at 0.05 (two-tailed).

Results

TF titration was performed using multiple dilutions of the TF preparation ranging from 1 : 17 (∼ 350 pmol L−1), to 1 : 34 000 (∼ 0.175 pmol L−1), and TF-free buffer serving as control. In each dilution step, 20 µL was added to whole blood from three healthy volunteers. t,MaxVel and CT prolonged with decreasing amount of TF. In healthy volunteers t,MaxVel, CT, and MaxVel changed approximating an ultimate time limit as shown in Fig. 2. Noteworthy, the buffer control experiment (Fig. 2) showed that even at very low concentrations, TF still influences the course of clot formation significantly. In patients with severe hemophilia A the t,MaxVel and the MaxVel values were also highly influenced by TF, even with a dilution of 1 : 30 600 (∼0.20 pmol L−1) (Fig. 3). With decreasing amounts of TF we observed a considerable increase in the interindividual variation, approximating the variation observed when no activator was employed (data not shown).

Figure 2.

Tissue factor titration in healthy individuals. Dynamic parameters of whole blood clot formation in three healthy volunteers following addition of tissue factor at final concentrations of ∼350 pmol L−1 to ∼0.175 pmol L−1 and a buffer control. (a) Mean and range of t,MaxVel with various concentrations of TF. (b) Mean and range of MaxVel with various concentrations of TF. (c) Mean and range of AUC with various concentrations of TF.

Figure 3.

Tissue factor titration in haemophiliacs. Characteristic velocity profiles of whole blood clot formation in a patient with severe haemophilia A following addition of tissue factor at final concentrations ranging from ∼1.75 pmol L−1 to ∼0.20 pmol L−1 and a buffer control. Velocity profiles with varying TF dilutions in severe hemophilia A whole blood: ∼1.75 pmol L−1 (black), ∼0.88 pmol L−1 (red), ∼0.58 pmol L−1 (green), ∼0.44 pmol L−1 (yellow), ∼0.35 pmol L−1 (blue), ∼0.25 pmol L−1 (purple), ∼0.20 pmol L−1 (light blue), buffer (orange).

Re-calcification with 20 µL 0.2 mol L−1 CaCl2 resulted in a Ca2+ level varying between 3.00 mmol L−1 and 3.27 mmol L−1 during the 120 min rest. The pH values changed from 7.29 to 7.37 in the re-calcified whole blood during 120 min. Respite times of 15, 30, 45, 60, 75, 90, 105, and 120 min were evaluated in three volunteers employing ∼0.35 pm TF as activator. The influence of different respite times of the citrate blood sample are shown in Fig. 4. During the first 30 min of respite t,MaxVel, CT, MaxVel, and CFT varied extensively. During this time period t,MaxVel, CT, and shortened while MaxVel increased. During the respite time interval from 30 min to 75 min there was no notable change in either parameter. Hence, we subsequently selected a respite time for the whole blood samples at 30 min.

Figure 4.

Influence of the length of respite time of citrated whole blood before thrombelastographic analysis. Dynamic parameters of whole blood clot formation in three healthy volunteers following varying respite times ranging from 0 to 120 min. (a) Mean t,MaxVel and range as a function of respite time of the whole blood sample. (b) Mean MaxVel and range as a function of respite time of the whole blood sample. (c) Mean AUC and range as a function of respite time of the whole blood sample.

The roTEG data from reference persons are given in Table 1. There was a significant higher MaxVel as well as a shorter CFT, CT, and, t,MaxVel in females compared to males with TF in a concentration of ∼0.35 pmol L−1. When Synthasil was used as the activating agent there was a tendency for females to express a brisker course of whole blood clot formation compared to males, but the difference was not statistically significant. The imprecision (CV%) was lower when employing the DyCoDerivAu™ derived parameters compared to use of the roTEG standard software for the CT (synthasil: CV = 5.0%; TF ∼0.35 pmol L−1 CV = 12.8%), CFT (synthasil: CV = 7.1%, TF ∼0.35 pmol L−1 CV = 17.4%) vs. t,MaxVel (synthasil: CV = 3.7%, TF ∼0.35 pmol L−1 CV = 9.9%) and MaxVel (synthasil: CV = 4.0%, TF ∼0.35 pmol L−1 CV = 7.3%) by the DyCoDerivAu™ software. The coefficients of variance for the AUC and the MCF displayed no difference between synthasil and TF ∼0.35 pmol L−1 series with values of CV at 2.3% and 3.0%, respectively.

Table 1. roTEG data from reference persons roTEG data in healthy reference persons
  Tissue factor (0.35 pmol L−1)Synthasil
Female (n= 30)Male (n= 30) Female (n= 30)Male (n= 30) 
  • *

    P < 0.05. NS, not significant; SD, standard deviation.

DyCoDerivAu™ parameters
 t, MaxVel (s)Average506.8552.4 * 256.0265.4NS
SD70.475.2 19.217.5 
 MaxVel (mm* 100 s−1)Average16.814.8 * 22.320.9NS
SD3.22.2 3.62.8 
 AUC (mm* 100)Average5951.95821.6NS6032.15955.4NS
SD507.7436.7 511.9468.8 
roTEG standard parameters
 CT (s)Average322.0354.2 * 121.4128.8NS
SD55.357.6 15.513.4 
 CFT (s)Average119.6141.2 * 62.167.2NS
SD28.026.2 13.512.2 
 MCF (mm* 100)Average6131.75946.7NS6356.56187.9NS
SD496.5372.9 469.3410.5 

The day-to-day variation was tested in two persons over five consecutive days. MaxVel varied between 10 and 14%, while t,MaxVel varied between 17 and 20%.

Figure 5(a–d) illustrates different characteristic profiles of whole blood clot formation recorded by employing TF at a concentration of ∼0.35 pmol L−1 as activator. Figure 5(a) illustrates velocity profiles of whole blood clot formation in a healthy volunteer, a patient on vitamin K antagonist treatment (INR = 3.1) owing to implantation of a mechanical heart valve, and a patient undergoing continuous infusion of heparin during extra corporal membrane oxygenation (heparin level @10 U anti-Xa/mL). Fig. 5(b) depict velocity profiles in a patient on vitamin K antagonist treatment (INR = 2.7) owing to implantation of a mechanical heart valve, and (c) a patient with severe hemophilia A and inhibitors, respectively, before and after addition or administration of rFVIIa. The patient with severe hemophilia A and inhibitors had a severely depressed MaxVel and a long t,MaxVel. In contrast, the patient on vitamin K antagonists only showed a modest depression of the MaxVel, but the t,MaxVel was prolonged. Following addition of rFVIIa, the t,MaxVel shortened and MaxVel increased to values inside the reference interval of healthy persons. Furthermore, in a severe hemophilia A patient with inhibitors, administration of rFVIIa (90 µg kg−1 bodyweight) injected every 2 h resulted in a cumulative effect on the MaxVel as well as on the t,MaxVel. In the latter case following the third dose of rFVIIa, the final FVIIc level reached ∼100 IU mL−1. For comparison, Fig. 5(d) shows the velocity profile captured in the same hemophilia patient's blood after ex vivo addition of 100 IU mL−1 rFVIIa.

Figure 5.

(a–d) Characteristic velocity profiles of whole blood clot formation in haemophilia and in anticoagulant therapy. Whole blood clot formation recorded employing ∼0.35 pmol L−1 of tissue factor as activator. (a) Black: Healthy person's velocity profile. Red: Patient on vitamin K antagonist (INR=3.1). Green: Patient on continuous heparin infusion during extra corporal membrane oxygenation [anti-Xa @10 U mL−1]. (b) Black: Patient on vitamin K antagonists before ex vivo addition of rFVIIa. Red: After ex vivo addition of 10 IU mL−1 rFVIIa; Green: after ex vivo addition of rFVIIa at 20 IU mL−1. (c) Velocity profiles of whole blood clot formation in a patient with hemophilia A with inhibitors. Velocity profiles repeated after three 2-hourly administration of rFVIIa during hip surgery. Black: before addition of rFVIIa. Red: After administration of first dose of rFVIIa 90 μg kg−1. Green: after administration of the second dose of rFVIIa 90 μg kg−1. Yellow: after administration of the third dose of rFVIIa 90 µg kg−1. (d) Ex vivo profiles of whole blood coagulation in the same patient as in (c). Black: Untreated severe haemophilia A with inhibitors. Red: Following ex vivo addition of rFVIIa at 100 IU mL−1.

Discussion

In the healthy individual, thrombin is generated from the interactions of several coagulation factors, cells, and platelets in the blood, and fibrin polymerization is the physiological consequence of the thrombin generation [13]. Thrombelastographic recording of the whole blood coagulation process is anticipated to indirectly reflect the course of thrombin generation. The appreciated importance of TF in thrombin generation has been the theoretical basis for employing activation with a diluted TF solution in our study. The pivotal TF titration studies revealed an apparent threshold phenomenon for MaxVel as well as for t,MaxVel in blood of healthy individuals. At a TF dilution of 1 : 17 000 (∼ 0.35 pmol L−1) the course of coagulation was distinctly different from the TF-free buffer control (Fig. 2). Choosing the same TF concentration, marked differences were found in the roTEG profiles of healthy volunteers compared to that of patients with severe hemophilia in whom we consistently found a seriously slowed and depressed clot formation profile. With high final concentrations of TF in the reaction system, the TEG profiles of hemophiliacs and of healthy people were almost identical. Moreover, using the selected dilution of TF, the total time of analysis was considered acceptable for routine clinical applications. By employing the very small amount of TF, the TEG coagulation signature was enhanced as compared to a TF-free system, still displaying distinct differences between healthy subjects and patients with hemophilia and those with reduced levels of vitamin-K dependent coagulation factors. To exclusively investigate TF dependent thrombin activation and clot formation, the contact pathway should be quenched by a corn trypsin inhibitor [14]. However, this was not adopted in our model assuming a minimal spontaneous activation in the all-plastic milieu.

TF activated coagulation as determined by thrombin generation has been investigated in platelet poor plasma as well as in platelet rich plasma, and automated methods have been devised to monitor the endogenous thrombin potential [15]. The dependency of thrombin generation in thrombelastographic parameters has not yet been investigated. However, the pattern of the new parameters introduced here, such as the MaxVel and t,MaxVel, display a remarkable degree of similarity between the endogenous thrombin potential (thrombogram [15]) and our thrombelastographic model.

Because of a pronounced variance in the TEG parameters within the first 30 min after venipuncture, all samples were rested for 30 min before recording. Another group of investigators suggested that analysis in citrated whole blood was safely executed from 1 to 8 h after the venipuncture [16]. However, Bowbrick et al. support our findings that a respite time of 30 min was sufficient [17]. Moreover, 30 min of rest improves the clinical feasibility of the test system.

Consistent with previous observations in native whole blood [18] a significant difference was detected between roTEG profiles of genders. In the TF ∼0.35 pmol L−1 based version, females expressed a higher MaxVel and a shorter t,MaxVel as compared to males. However, when tested in the Synthasil activated model, this difference was no longer significant. At this time, we have no clear explanation to the sex dependent relationship.

Reportedly, rFVIIa has been an efficient hemostatic tool in control of bleeding in hemophiliacs with inhibitors as well as in some other kinds of coagulopathies [19]. However, a method for biochemical monitoring of the efficacy of rFVIIa ex vivo has not yet been established. As previously forwarded [20], thrombelastography based on low TF activation might be a suitable method for monitoring of hemostatic intervention with rFVIIa [21]. rFVIIa has also been proposed as a hemostasis promoting agent in bleedings caused by vitamin K antagonists as illustrated by studies in volunteers, animal studies, and case reports [22–25]. From our graphs (Fig. 5b) we suggest that rFVIIa might possess the capacity to promote hemostasis in vitamin K antagonist treated individuals.

The implementation of the dynamic parameters as well as their graphical display facilitates the interpretation of the thrombelastogram and improves the resolution of smaller differences amongst samples processed. Additionally, the novel coagulation software improves the precision of double determinations as assessed by the coefficient of variation (CV%) of double determinations. A likely explanation for this could be a more well-defined peak of the velocity curve as compared to traditional deflection angle estimate in the original TEG profile.

Our data suggest that the profile of whole blood coagulation by thromboelastography, as an indirect measure of thrombin generation, may provide extensive information on hemostasis at the bed-side level in patients with severe hemostasis problems. In addition, the presented method seems to allow for improved monitoring of the efficacy of rFVIIa in patients with severely impaired hemostasis. Ongoing studies will aim at exploring hemostasis in patients with various abnormalities to predict the possible effects of interventive measures by ex vivo procedures.

Acknowledgements

We wish to thank the voluntary donors of the Blood Bank at Skejby Sygehus for their participation and co-operation. Further, we are indebted to the staff of the Department of Cardiothoracic and Vascular Surgery for assisting us with measurements of pH and ionized calcium. Furthermore, we would like to acknowledge the help of Niels Trolle Andersen, the Department of Bio-Statistics for consulting on the statistical work.

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