 renal and hepatic function. For all 8 volunteers, baseline measurements for fibrinogen (160–400 mg/dL), platelet count (150–400 G/L) and hematocrit (36%–46%), as well as baseline values of the TEG® (clotting time [CT], 100–240 s; clot formation time [CFT], 35–110 s; maximum clot firmness [MCF], 53–72 mm for the INTEM® test) were in the respective physiological ranges.
The test samples were analyzed by means of a modified TEG® coagulation analyzer (ROTEM®; Pentapharm CO, Munich, Germany) based on the TEG® system described by Hartert (11,12). In the ROTEM® system the sensor pin is guided by a ball-bearing system that makes it less susceptible to movement and vibration. Activation of the test samples accelerates the measurement process and enhances the reproducibility in comparison with native TEG® measurement (13). The thrombelastogram is characterized by specific variables (Fig. 1). The CT corresponds to the reaction time in a conventional TEG® and describes the initiation of clot formation. The CFT is equivalent to the coagulation time and reflects the dynamics of the coagulation process. It is defined as the time from the onset of clotting until a clot firmness of 20 mm is reached. Finally, the MCF refers to the maximum amplitude and represents the quality of the clot.
The ROTEM® thrombelastogram (TEG®) is characterized by specific parameters. The “coagulation time” (CT) corresponds to the reaction time (r) in a conventional TEG® and describes the initiation of coagulation. The “clot formation time” (CFT) is equivalent to the coagulation time (k) and illuminates the dynamic of the coagulation process. Finally, the “maximum clot firmness” (MCF) refers to the maximum amplitude and represents the quality of the clot.
In the first experimental series, we diluted samples of citrated whole blood with either NaCl 0.9% (Fresenius, Bad Homburg, Germany) or HES 6% 130/0.4 (Voluven®, Fresenius). Both solutions were supplemented with sodium citrate in a 1:10 proportion before use to attain the same citrate concentration in all test samples. For each experiment, 300 μL of whole blood, blood-NaCl 0.9%, or blood-HES 6% dilution was analyzed. Dilutions of 0% (control), 20%, 40%, 60% and 80% were prepared (volume proportion of NaCl 0.9% or HES 6% in the tested dilutions).
In the second experimental series, we used 60% dilution of whole blood to analyze the influence of application of fibrinogen concentrate, platelet concentrate, or both together on ROTEM® variables. We diluted 1.2 mL of citrated whole blood with either 1.8 mL HES 6% or with 1.8 mL NaCl 0.9% and added either 200 μL of fibrinogen concentrate (Hemocomplettan® HS, ZLB Behring, Bern, Switzerland) with a stock concentration of 20 mg/mL, 100 μL of a commercial platelet apheresis concentrate, or both components. The chosen dosages of platelets and fibrinogen corresponded to an application of 6 g fibrinogen or one platelet apheresis concentrate (150 mL) to a total blood volume of 5000 mL. One sample of citrated whole blood remained undiluted, serving as a baseline measurement (control). Two others were diluted with either HES 6% or NaCl 0.9% without addition of fibrinogen or platelets to serve as dilution controls.
According to the manufacturer’s instructions, citrated whole blood was recalcified with buffered CaCl2 (0.2 M, 20 μL) and the intrinsic pathway activator ellagic acid (20 μL) was added (INTEM®, Pentapharm, Munich, Germany). In a separate measurement, the effect of platelets on the clot formation was blocked by the addition of cytochalasin D (FIBTEM®, Pentapharm). The resulting clot firmness was based only on the polymerized and cross-linked fibrin. All ROTEM® measurements were performed at a test temperature of 37°C.
Hematocrit and platelet count were measured by an automated analyzer (Sysmex SE 9000B; Sysmex Europe, Norderstedt, Germany). Fibrinogen concentration was determined with the Clauss method.
To display the effects of dilution and restitution of fibrinogen on blood clot formation, scanning electron microscopy was performed on one set of blood clots. After measurement by ROTEM®, blood clots were extracted from the test cup and immediately placed in 3% glutaraldehyde. After 2 hours of fixation, glutaraldehyde was diluted with distilled water to a concentration of 1% for further storage overnight. The next day, the fixed clots were repeatedly washed in phosphate buffered NaCl 0.9% and then postfixed for another 2 h in 2% osmium tetroxide. After several washing procedures and dehydration in a graded series of ethanol (30%–100%), the samples were dried, spattered with gold, and examined with a JEOL scanning electron microscope (JEOL Ltd., Tokyo, Japan).
For statistical analysis of the changes induced by hemodilution, and comparison of the two intervention groups, one-way repeated measures analysis of variance was performed with post hoc procedures according to the Student-Newman-Keuls test for all pairwise testing and Bonferroni/Dunn’s test for testing versus a control group. All variables are expressed as mean ± sd.
Increasing dilution led to dose-dependent impairment of clot formation of whole blood. The effects were more pronounced with HES 6% than with NaCl 0.9%. Figure 2 shows the effects on TEG® variables. The normal range for the CT is ≤240 s. An extension to ≥400 s is a sign of severe impairment of initiation of the coagulation process. The CFT is normally ≤110 s. A CFT ≥250 s indicates a major disorder of clot formation. Healthy individuals show an INTEM® MCF between 53 mm and 72 mm. A reduction of the MCF to ≤40 mm is a sign of strongly impaired hemostasis. For the FIBTEM® a MCF of ≤8 mm indicates a lack of fibrinogen or a disturbance of the fibrin polymerization. In our dilution series, only 80% dilution with HES 6% resulted in a severe impairment of the CT. Thus, the initiation of clot formation seems to be quite resistant to dilution effects. In contrast, CFT and MCF in the INTEM® test were already strongly impaired after 40% dilution with HES 6%, whereas 60% dilution with NaCl 0.9% was necessary to induce the same effect. Even more pronounced was the situation concerning the MCF in the FIBTEM® test, which relies only on fibrin clot formation and fibrin polymerization. Twenty percent dilution with HES 6% led to severe impairment, and 40% dilution even resulted in a complete inhibition of clot formation. In the case of NaCl 0.9%, 60% dilution scarcely disturbed clot formation. However, 80% dilution inhibited it severely. With respect to CFT and MCF in each of the 20%–60% dilutions, HES 6% impaired coagulation significantly stronger than an equivalent dilution with NaCl 0.9%.
Effect of progressive in vitro hemodilution (20%, 40%, 60%, 80%) with NaCl 0.9% (dashed lines) and HES 6% (solid lines) on blood coagulation as assessed by thrombelastography. Coagulation time, clot formation time, and maximum clot firmness (MCF) were measured by the INTEM test (clot formation after intrinsic activation), and MCF was measured additionally by the FIBTEM test (selective fibrin clot formation). Data are mean ± sd, n = 8. *P < 0.05 versus the control of the same group; # P < 0.05 versus the other group for the same intervention. The dashed horizontal line indicates the critical value for severe impairment of coagulation.
A dilution grade of 60% was chosen for the intervention series, as this led to a significant impairment of coagulation for both diluents. The results of these experiments are depicted in Figure 3. Supplementation with fibrinogen, platelets, or both reduced CT significantly in the case of HES 6%, whereas only the combined application resulted in a significant shortening of CT after dilution with NaCl 0.9%. However, this statistically significant improvement would seem to be of minor clinical relevance, as it only represents a return to normal from a mildly extended CT. In contrast, CFT was massively prolonged after dilution with HES 6% but not critically by NaCl 0.9%. In the case of HES 6%, only the application of platelets, but not of fibrinogen, was able to reduce the CFT significantly. The combination of both components was not superior to the addition of platelets alone. CFT impairment after dilution with NaCl 0.9% improved significantly after the application of fibrinogen or platelets, but no additional effect was detectable when both components were added together. A 60% dilution of whole blood deteriorated MCF in the INTEM® test to a statistically significant and clinically relevant extent. The changes were significantly more pronounced for HES 6% than for NaCl 0.9%. In the presence of NaCl 0.9% only the application of fibrinogen was able to improve the INTEM® MCF to a clinically relevant extent. The combination with platelets was not superior to the sole addition of fibrinogen. In contrast, after dilution with HES 6% no intervention improved the MCF in the INTEM® test to clinically desirable values. Qualitatively identical results were obtained for the measurement by the FIBTEM® test. The addition of fibrinogen reconstituted the fibrin clot firmness fully in the presence of