The molecular weight of hydroxyethyl starch (HES) solutions may be an important characteristic in determining their effects on blood coagulation during hemodilution [1-3]. We designed the present study to investigate the effects of a recently developed HES solution with a molecular weight of 130 kD (HES 130) compared with the currently widely used HES solution with a molecular weight of 200 kD (HES 200) on in vitro blood coagulation using thrombelastography (TEG) . To differentiate between a hemodilutional effect per se and an intrinsic colloidal effect of different HES solutions, we simultaneously compared TEG variables of native (i.e., undiluted) blood, blood diluted with 0.9% saline, and blood diluted with HES 130 and HES 200 .
With ethics committee approval and written, informed consent, 80 patients scheduled for various surgical procedures were enrolled in the study. Exclusion criteria were a history of preoperative coagulation disorders, oral anticoagulation within 5 days before surgery, treatment with heparin and/or acetylsalicylic acid (aspirin) within 5 days before surgery, use of nonsteroidal antiinflammatory drugs within 24 h before surgery, history of renal dysfunction or increased serum creatinine levels (>120 [micro sign]mol/L), and history of liver dysfunction or increased plasma levels of aspartate aminotransferase (>50 U/L) or alanine aminotransferase (>50 U/L). A standard thrombosis prophylaxis of 3000 U of low molecular weight heparin subcutaneously was allowed.
We investigated the effects on in vitro blood coagulation of the following two solutions: 6% HES 200/0.5 (HAES-steril 6%; Fresenius AG, Bad Homburg, Germany)-a HES solution with a mean molecular weight of 200 kD, a degree of substitution of 0.5, and a C2 to C6 ratio of 5.1 (the C2 to C6 ratio describes the ratio of hydroxyethyl groups substituted at the C2 and C6 positions of the glucose molecule)-and 6% HES 130/0.4 (Hydroxyethylstarke 130/0.46%; Fresenius AG)-a HES solution with a mean molecular weight of 130 kD, a degree of substitution of 0.4, and a C2 to C6 ratio of 11.2.
Blood samples were randomly allocated to a 30% and a 60% in vitro hemodilution group. In each blood sample, blood coagulation was assessed simultaneously by using TEG in native (i.e., undiluted) blood and after in vitro hemodilution to the same degree (either 30% or 60% hemodilution) with HES 200/0.5, HES 130/0.4, and 0.9% saline solution.
As we learned from a previous study , CaCl2 and sodium bicarbonate had to be added to the HES solutions and to the 0.9% saline solution to maintain physiological Ca2+ and pH levels in the in vitro hemodiluted blood because there are no homeostatic mechanisms in vitro. The prehemodilution values found to result in physiological Ca2 and pH levels after hemodilution were 0.9% saline solution (Ca2+ 1.10-1.16 mmol/L, pH 7.30-7.65), HES 130/0.4 (Ca2+ 1.13-1.18 mmol/L, pH 7.30-7.65), and HES 200/0.5 (Ca2+ 1.13-1.18 mmol/L, pH 7.30-7.65). Without adjustment, Ca2+ and pH levels would decrease after in vitro hemodilution and might impair blood coagulation [6,7].
In each patient, four TEG tracings (CTEG 3000; Hemoscope, Morton Grove, IL) were performed simultaneously: native blood and blood hemodiluted to the same degree (to either 30% or 60% hemodilution) with 0.9% saline, HES 130/0.4, and HES 200/0.5. A 10-mL blood sample was collected into two 5-mL polypropylene syringes (for ease of blood withdrawal) from freshly inserted peripheral venous, central venous, or radial arterial catheters. The first 2 mL was discarded to avoid coagulation enhancement through tissue factors released by insertion of the needle into the vessel. From the remaining 8 mL, the appropriate amount of blood was added by reverse pipetting to prewarmed (37[degree sign]C) 0.9% saline, HES 130/0.4, and HES 200/0.5 solutions in polypropylene tubes. The solutions were prewarmed to prevent the interference of hypothermia with coagulation . The native and hemodiluted blood samples were mixed five times by filling and half-emptying the pipette. Three minutes after blood withdrawal, 1 mL of each sample was added to tubes containing 1% celite. After inverting the tubes five times, 360 [micro sign]L of each celite-activated sample was pipetted into the prewarmed plastic TEG cups. The blood samples were covered with mineral oil to prevent evaporation, and the four TEG traces (native blood and blood hemodiluted with 0.9% saline, HES 130/0.4, and HES 200/0.5) were simultaneously started precisely 6 min after blood withdrawal. During the entire measurement process, the temperature of the TEG was kept at 37[degree sign]C. To control the level of hemodilution and of Ca2+ and pH, hemoglobin, blood gases, and electrolytes were measured in the remainder of the native and hemodiluted blood samples (BG-Electrolytes and CO-Oximeter; Instrumentation Laboratory, Lexington, MA).
We used TEG to assess blood coagulation. The basic functional principle of TEG has been described previously [4,5,9-12]. The following TEG variables were assessed: reaction time (r; normal range 10-14 mm), coagulation time (k; normal range 3-6 mm), maximal amplitude (MA; normal range 59-68 mm), angle alpha (normal range 54-67[degree sign]), and clot lysis, described by Ly30 and Ly60 (normal range <7.5% and <15%, respectively) . To assess overall coagulation, a TEG coagulation index (CI) including all individual TEG variables was calculated for celite-activated blood (CI = -0.3258 [center dot] r - 0.1886 [center dot] k + 0.1224 [center dot] MA + 0.0759 [center dot] alpha- 7.7922) [11,13]. The normal range is from -2.0 to 2.0. Outside this range, a higher positive value indicates hypercoagulability and a lower negative value indicates hypocoagulability.
Characteristics of patients donating blood that was subsequently hemodiluted by 30% or 60% were compared using unpaired t-tests (Statview 4.51; Abacus Concepts, Inc., Berkeley, CA). The effect of 30% or 60% hemodilution, compared with the native blood, was assessed using paired t-tests with Bonferroni adjustment. Variables of hemodiluted blood (30%, 60%) with HES 130/0.4 and HES 200/0.5 were analyzed using repeated-measures analysis of variance with Greenhouse-Geisser correction (Superanova 1.11; Abacus Concepts, Inc.). P < 0.05 was considered statistically significant. Data are presented as mean +/- SD or mean +/- SEM as appropriate.
The 30% and 60% hemodilution groups were similar regarding demographic characteristics (Table 1), baseline data (Table 2), and TEG variables (Table 3). The only difference found a marginally higher serum creatinine level in the 60% hemodilution group.
Hemoglobin concentration decreased similarly after in vitro hemodilution to 30% or 60% with 0.9% saline, HES 130/0.4, and HES 200/0.5 (Table 2). The Ca (2+) concentration remained stable during hemodilution with all three solutions. pH also remained stable except for a minimal increase at 30% hemodilution with 0.9% saline and a slight decrease at 60% hemodilution with both HES solutions, although both remained within physiologic limits (Table 2).
Blood coagulation was increasingly compromised by progressive in vitro hemodilution with both HES solutions. r and k increased, and MA, angle alpha, and CI decreased progressively with increasing degree of hemodilution (Figure 1). Among the variables, no significant difference was found between blood hemodiluted with HES 130/0.4 and blood hemodiluted with HES 200/0.5 (Figure 1). Clot lysis 30 min after MA (Ly30) increased significantly only after 30% hemodilution with HES 200/0.5. Ly60 increased significantly after 30% hemodilution with both HES solutions. Clot lysis after 60% hemodilution showed no difference from values for native blood. Again, there was no significant difference between HES 130/0.4 and HES 200/0.5 (Figure 1).
Thirty percent hemodilution with 0.9% saline induced a significant decrease in r and MA and a significant increase in angle alpha (Table 3). There was no change in k and CI compared with native blood. Ly30 was unchanged, and Ly60 decreased slightly. Sixty percent hemodilution with 0.9% saline induced a significant increase in r and k and a significant decrease in MA, angle alpha, and CI. Both Ly30 and Ly60 decreased compared with native blood (Table 3).
To evaluate the intrinsic effect of the two HES solutions on blood coagulation, we analyzed the difference between blood diluted with 0.9% saline and blood diluted with HES (expressed as absolute difference of the values, i.e., values for HES diluted blood minus the values for saline diluted blood). Hemodilution with both HES solutions increased r and k and decreased MA and angle alpha more than hemodilution with 0.9% saline (Figure 2). Changes with 60% hemodilution were greater than with 30% hemodilution (P < 0.001) except for r and Ly30. Among the variables, there was no significant difference between HES 130/0.4 and HES 200/0.5 (Figure 2).
We found no difference in the in vitro blood coagulation compromising potency between HES 130/0.4 and HES 200/0.5.
HES molecules are usually characterized by their mean molecular weight, their degree of substitution (defined as the average number of hydroxyethyl groups per glucose moiety), and their C2 to C6 ratio, which describes the ratio of hydroxyethyl groups substituted at the C2 and C6 positions of the glucose molecule. These structural aspects of HES molecules also modulate the influence on blood coagulation . The greater the molecular weight [1,2,14,15], the higher the degree of substitution [2,16,17] and the C2 to C6 ratio [2,18], the more a particular HES solution will compromise coagulation.
Despite different molecular weights of 130 kD and 200 kD, we found no difference between the effect of HES 130/0.4 and HES 200/0.5 on in vitro blood coagulation in the present study. The difference in mean molecular weight, however, was not the only difference between the two HES solutions. The degree of substitution was also minimally different (0.4 vs 0.5), and, more importantly, there was a significant difference in the C2 to C6 ratio. HES 130/0.4 has a higher C2 to C (6) ratio (11.2) than HES 200/0.5 (5.1). Because a high C2 to C6 ratio is associated with an exaggerated blood coagulation compromising effect , it is conceivable that the effects of a lesser mean molecular weight and a higher C2 to C6 ratio of HES 130/0.4 resulted in a blood coagulation-compromising potency similar to the higher molecular weight but in a lower C2 to C6 ratio of HES 200/0.5.
In the present study, all TEG values for native blood were slightly hypercoagulable (Figure 1, Table 3) compared with normal ranges. This relative hypercoagulability may be explained by different batches of celite used in this and the previous study . Celite is a suspension of silica particles that is very difficult to produce consistently at any given concentration (E. Cohen, Hemoscope, Morton Grove, IL, personal communication, 1998). It is therefore important to use one batch of celite for the entire duration of a particular study, as was the case in this and the previous study .
Moderate hemodilution with crystalloids facilitates blood coagulation in vitro [4,5] and induces mild hypercoagulability in vivo [9,19,20] even after the infusion of as little as 1 L of 0.9% saline . We observed this hypercoagulability in the present study after 30% hemodilution with 0.9% saline, as evidenced by a decrease in r and an increase in the angle alpha (Table 3). In contrast, MA decreased slightly, and CI, the overall TEG measure of blood coagulation, tended to increase. Blood coagulation facilitation due to hemodilution with crystalloids thus was flawed in the present study, most likely because the relative hypercoagulability found in native blood related to a somewhat more active batch of celite used in the present study. At 60% hemodilution with 0.9% saline, r and k increased and MA, angle alpha, and CI decreased, indicative of compromised blood coagulation with advanced hemodilution with crystalloids, as has been observed previously [4,5]
Extrapolating these findings to clinical practice is very difficult. However, given the high sensitivity of TEG to detect even minor changes in blood coagulation, it is unlikely that clinically relevant differences between HES 130/0.4 and HES 200/0.5 will be found when these substances are used intraoperatively. However, future in vivo studies are necessary for definitive validation.
Interestingly, hemodilution with 0.9% saline progressively decreased clot lysis both at 30 and 60 min (Table 3), as described previously . Analyzing an intrinsic HES effect on clot lysis thus mandates compensating for these changes by hemodilution per se. By doing so, a progressive augmentation of clot lysis during hemodilution with either HES solution investigated becomes apparent (Figure 2). HES thus seems to increase clot lysis, although within normal limits. Again, there was no difference found between HES 130/0.4 and HES 200/0.5.
In conclusion, the present study demonstrates that HES 130/0.4 and HES 200/0.5 compromise in vitro blood coagulation to the same degree.
1. Strauss RG, Stansfield C, Henriksen RA, Villhauer PJ. Pentastarch may cause fewer effects on coagulation than hetastarch. Transfusion 1988;28:257-60.
2. Treib J, Haass A, Pindur G. Coagulation disorders caused by hydroxyethyl starch. Thromb Haemost 1997;78:974-83.
3. Treib J, Haass A, Pindur G, et al. Avoiding an impairment of factor VIII:C by using hydroxyethyl starch with a low in vivo molecular weight [letter]. Anesth Analg 1997;84:1391.
4. Ruttmann TG, James MF, Viljoen JF. Haemodilution induces a hypercoagulable state. Br J Anaesth 1996;76:412-4.
5. Egli GA, Zollinger A, Seifert B, et al. Effect of progressive haemodilution with hydroxyethyl starch, gelatin and albumin on blood coagulation. Br J Anaesth 1997;78:684-9.
6. Dunn EL, Moore EE, Breslich DJ, Galloway WB. Acidosis-induced coagulopathy. Surg Forum 1979;30:471-3.
7. Bithell TC. Role of metal ions. In: Lee GR, Bithell TC, Foerster J, et al., eds. Wintrobes clinical hematology. Philadelphia: Lea & Febiger, 1993:587.
8. Gubler KD, Gentilello LM, Hassantash SA, Maier RV. The impact of hypothermia on dilutional coagulopathy. J Trauma 1994;36:847-51.
9. Tuman KJ, Spiess BD, McCarthy RJ, Ivankovich AD. Effects of progressive blood loss on coagulation as measured by thrombelastography. Anesth Analg 1987;66:856-63.
10. Mallett SV, Cox DJ. Thrombelastography. Br J Anaesth 1992;69:307-13.
11. Caprini JA, Zuckerman L, Cohen E, et al. The identification of accelerated coagulability. Thromb Res 1976;9:167-80.
12. Ruttmann TG, James MFM, Aronson I. In vivo investigation into the effects of haemodilution with hydroxyethyl starch (200/0.5) and normal saline on coagulation. Br J Anaesth 1998;80:612-6.
13. Sharma SK, Philip J, Wiley J. Thromboelastographic changes in healthy parturients and postpartum women. Anesth Analg 1997;85:94-8.
14. Treib J, Haass A, Pindur G, et al. Influence of intravascular molecular weight of hydroxyethyl starch on platelets. Eur J Haematol 1996;56:168-72.
15. Treib J, Haass A, Pindur G, et al. Influence of low molecular weight hydroxyethyl starch (HES 40/0.5-0.55) on hemostasis and hemorheology. Haemostasis 1996;26:258-65.
16. Treib J, Haass A, Pindur G, et al. All medium starches are not the same: influence of the degree of hydroxyethyl substitution of hydroxyethyl starch on plasma volume, hemorheologic conditions, and coagulation. Transfusion 1996;36:450-5.
17. Treib J, Haass A, Pindur G, et al. Increased haemorrhagic risk after repeated infusion of highly substituted medium molecular weight hydroxyethyl starch. Arzneimittelforschung 1997;47:18-22.
18. Treib J, Haass A, Pindur G, et al. HES 200/0.5 is not HES 200/0.5: influence of the C2/C6 hydroxyethylation ratio of hydroxyethyl starch (HES) on hemorheology, coagulation and elimination kinetics. Thromb Haemost 1995;74:1452-6.
19. Janvrin SB, Davies G, Greenhalgh RM. Postoperative deep vein thrombosis caused by intravenous fluids during surgery. Br J Surg 1980;67:690-3.
20. Ng KF, Lo JW. The development of hypercoagulability state, as measured by thrombelastography, associated with intraoperative surgical blood loss. Anaesth Intensive Care 1996;24:20-5.