Hydroxyethyl starch (HES) solutions are widely used to replace intravascular volume. Dilution by crystalloids or colloids and platelet dysfunction are known causes of perioperative bleeding disorders (1). Because HES solutions have varying physicochemical properties with regard to molecular weight and degree of hydroxylation, they may interfere with the coagulation system differently: the higher the degree of substitution, the molecular weight, and the C2:C6 ratio, the slower the elimination kinetics (2). So, dilutional effects, effects on von Willebrand factor complexes, and effects on platelets are different (3). In contrast, data on elimination characteristics of different HES solutions (4) and data on the interference of HES with the early stage of coagulation are scarce.
We investigated the influence of moderate and profound in vitro hemodilution with different widely used HES solutions (70.000/0.5, 200.000/0.5) and a recently developed preparation (130.000/0.4) on blood coagulation. A SONOCLOT analysis (SCT; Sonoclot II Coagulation and Platelet Function Analyzer, Sienco Co., Morrison, CO) and standard laboratory tests were used for comparison. To differentiate between the dilutional and pharmacological effects of the HES solutions, the test results of the lactated Ringer’s solution group (RL) were taken as a reference.
After institutional ethical approval, 12 healthy volunteers without any recent drug intake donated 25 mL of blood for routine analysis, and an additional 2 mL for SCT, at five different times. Study inclusion criteria were male, nonsmoker, and no history of chronic or acute diseases and medication.
In order to minimize intrapersonal variability, the donation of the blood samples for the SCT was performed within 8 h by using a syringe technique. To avoid a thromboplastin and tissue factors contamination, the first specimen was aspirated and discarded (5). The blood for SCT was not stored. The SCT was started within 3 min after venous cannulation. The blood samples for routine variables were taken at once and stored for 3 h. Coagulation was prevented by citration (except the SCT cuvettes), and recalcification for analysis was done automatically in the coagulation lab. For all specimens except for those for SCT analyses, the time intervals between drawing of blood specimens and performing the routine analysis were equal in all dilution groups.
HES (molecular weight/degree of hydroxylation/concentration/C2:C6 hydroxyethyl ratio, HES 70.000/0.5/6%/3.2; Pharmacia & Upjohn Co., Erlangen, Germany; HES 130.000/0.4/6%/11.2 and HES 200.000/0.5/6%/4.6–5.2; Fresenius Co., Bad Homburg, Germany) and RL were added to the test tubes containing the blood from the volunteers. RL was used because of the balanced metabolism after rapid infusion compared with saline (6). Sample 1 (no dilution) was used for baseline values; Sample 2 was diluted to 33% (1/3 HES or RL and 2/3 blood); Sample 3 was diluted to 66% by using the test solutions (1/3 blood and 2/3 HES or RL). In the same manner, the blood in the SCT cuvette was either undiluted (Sample 1) or diluted to 33% (Sample 2) or 66% (Sample 3). Dilution was performed by volume, not weight. After dilution of the blood in the cuvette (total volume 400 μL), the specimens were measured according to the manual of the SCT machine.
The following laboratory tests were performed in the three samples: hematocrit, fibrinogen, red cell and platelet count, and activated partial thromboplastin time (aPTT). With the SCT machine, we investigated activated clotting time (ACT), clot rate (CR), time to peak (TTP), clot signal at peak analysis (maximal clot signal [MCS]), and prevalence of clot retraction.
The principle of the SCT is the registration of the difference of viscoelastic properties of a forming blood clot (7). The SCT uses impedance difference over time caused by clotting in a fresh whole blood sample, which is oscillated by a vibrating transducer (200 Hz, distance 1 μm, temperature 37°C) (8). The test cuvette contains 5 mg of a celite activator. Several measurements are performed (7). The ACT is the onset time of the beginning of fibrin formation. CR is the rate of fibrin formation from fibrinogen (primary slope [R1]). After primary fibrin formation, as measured by the CR, there is a variable inflection point measuring the start of fibrin retraction, which is activated by platelets. The second slope (R2), especially the peak value, represents the completion of clot formation, including all necessary portions of plasmatic and cellular clot formation. The MCS represents fibrinogen concentration, and TTP is the value of the speed of fibrin formation. The decline (R3) after MCS is caused by squeezing serum out of the clot matrix, which is induced by platelets. The number of available platelets as well as platelet function, determines this effect. Platelet dysfunction is seen by the lack of an inflection point between R1 and R2, a prolongation of the time to peak and a missing downslope after the maximal peak R3 (7). A positive retraction was defined as a downslope after the MCS, with a minimal decrease of 10 scale units. Further decrease (after the peak) is caused by clot lysis, which is rarely observed during standard SCT. Detailed methodology is discussed in a previous publication (9).
To calculate any differences between the coagulation results, nonparametric analysis of variance and adequate post hoc tests were used as appropriate by using Statistica 5.1 (StatSoft Inc., Tulsa, OK). Qualitative data were analyzed by using the nonparametric Kruskal-Wallis test with the Monte Carlo Setting with StatXact 3.0.2 (Cytel Software Corporation, Tulsa, OK). Data were presented as mean ± SD. Outliers and extremes were excluded from statistical analysis. For graphical presentation, box plots were chosen where the box represents 25%–75% of the entire data. Additionally, nonoutlier ranges, outliers, extremes, and median were given. They were defined mathematically. A P value below 0.05 was considered significant.
Routine laboratory variables were affected equally by the tested solutions, except for the aPTT (Tables 1 and 2).
The coagulation process was analyzed by the SCT in the following way: the liquid phase of coagulation (ACT) was affected by the 33% and 66% hemodilution. All 33% diluted samples showed a slight activation of the onset of coagulation, which was most visible in the RL group. The 66% diluted samples prolonged the ACT with a minimal effect in the RL group. Significant differences were found between the distinct diluted samples. The baseline ACT did not differ significantly to the variables in the 33% diluted group (Fig. 1).
Performing hemodilution reduced the CR significantly in all groups compared with the baseline variables. Minimal changes were observed in the RL group. Even in the 66% diluted samples, the CRs in the RL group were comparable with the other 33% diluted samples (Fig. 2).
Clot maturation and speed of maturation as measured by TTP was nearly unaffected by HES 130.000 and RL 33% diluted solutions, this was also visible in the more highly diluted samples. All other solutions showed significant adverse effects on clot maturation, which most pronounced with HES 70.000. HES 70.000 more than doubled the TTP, regardless of the degree of dilution. Significant differences were found between the 66% diluted groups and the baseline variables (Fig. 3).
The CR at peak was nearly unaffected by HES 130.000. Even in the 66% diluted sample, the variables were comparable to the baseline values. Significant differences were found between the RL and the HES 130.000 groups (Fig. 4).
The clot retraction was better in the HES 130.000 33% diluted group compared with the other HES solutions and compared significantly better to HES 200.000. HES 200.000 showed the least retraction (Table 3).
HES solutions have varying physicochemical properties with regard to molecular weight and degree of hydroxylation (2). This influences elimination kinetics, but also affects coagulation by the volume effect or effect on specific factors and platelets (2,3).
The tested HES preparations showed different coagulation patterns, as measured by the SCT compared with baseline values. Regardless of the degree of dilution, HES 130.000 affected fewer coagulation variables compared with HES 70.000 and 200.000, as measured by the SCT. Dilutional effects were identified by comparison of the HES groups with the samples diluted with RL. Routines laboratory variables were not able to identify any specific effects of the tested HES solutions, except for the aPTT.
HES solutions differ widely with regard to their pharmacokinetic and dynamic effects. There are few data on the clinical properties of short-term use of HES solutions. Medium molecular HES 200.000/0.62 preparations have been shown to interfere with coagulation significantly (drop of factor VIII and cofactors) when used for 10 days (4). This effect was attributed to the relatively high degree of substitution, but may also be a result of the duration of application. Other investigators found a relatively fast recovery of coagulation factors after a short-term application of HES 200.000/0.6 (10). Low molecular HES (40.000/0.5) was investigated during long-term application with no negative effect on coagulation (11). The molecular weight of the HES solution and the degree of substitution determine the anticoagulative properties of the HES solutions (2,12). Additionally, the C2:C6 substitution ratio is an important factor (13). We found similar effects on the coagulation system between HES 70.000 and 200.000, which were both substituted with 0.5, but with a different C2:C6 ratio. The reduced anticoagulative properties of the HES 130.000 may be caused by a better proportion of molecular weight and degree of substitution, with which a more pronounced difference between the HES 130.000 and 200.000 would be expected. This is probably compensated by the higher C2:C6 ratio of the HES 130.000. Our in vitro results preferring HES 130.000 are in concordance with some clinical studies that found fewer interactions with the coagulation between HES 130.000/0.4 and HES 200.000/0.5 (14). Slightly diverging in vitro results were published recently, but the differences may be explained by the use of different techniques, such as the use of thrombelastography versus SCT and adjustment of electrolytes and pH (15). In the present study, Ca2+ concentration and pH did not differ within the HES groups. The pH ranged from 7.31 to 7.41. The Ca2+ concentrations were significantly higher in the RL group compared with the HES groups (33%/66% dilution: 1.31–1.35 vs 0.4–0.8 mmol/L).
A slight activation of early coagulation was seen with all solutions, especially with RL, which can be interpreted as an activation of the initial coagulation cascade (16). This activation of coagulation was not observed during the later clot formation process. In our investigation, this effect was seen for all HES preparations, as found by other authors with thromboelastography (17,18). When the samples were diluted to 66%, this “hypercoagulability” was no longer observed in this early stage of clot formation. This phenomenon has been described for crystalloids as an early hypercoagulability (17). Recently, this effect was specified by the detection of an disproportional decline of antithrombin compared with the baseline after the administration of crystalloids (19).
During the clot maturation, a clot retraction can be seen. This retraction strengthens the mechanical properties of the clot. Clot retraction is caused by squeezing the serum out of the clot. HES 130.000, as well as RL, affected the maturation process significantly less compared with the other HES solutions. Changes in the clot morphology have been also described for dextran (20). Reasons for the described differences might be the different molecular attraction between fibrin molecules and the applied solution (21). Another explanation might be changes in the polymerization process of fibrin. HES changes the fibrin fiber mass:length ratios and clot elastic modulus, which changes tensile properties of the clot (22,23). An absent retraction indicates a disturbance in the natural maturation process of a clot, which is normally caused by the force development induced by platelets (23). The clinical impact of these findings is unclear.
Our study has certain limitations:in vitro assays miss physiological reactions during progressive hemodilution, such as recruitment of additional resources of the coagulation system. Another disadvantage is the change of Ca2+ concentration by dilution with the tested solutions, especially when RL was used instead of saline for comparison. The elevated Ca2+ concentrations in the RL group compared with the HES groups may have lead to larger differences. However, surgical stress, tissue damage, and/or endothelial injury and their effect on coagulation are eliminated in an in vitro model (18). Another limitation is the use of the SCT. Despite its wide clinical use, there are very few publications validating the test results of the SCT with other coagulation tests (7,24). As coagulation variables are dynamic, especially in the perioperative setting, extrapolation of our data to in vivo effects are difficult. Another methodological problem consists of the use of a celite activator. The available activator was primarily used in cardiac surgery to measure ACT and, so, may mask some slight differences between the test samples. Recently, a less potent activator was investigated but not available during the study.
In conclusion, performing in vitro hemodilution HES 130.000 and 200.000 offers several advantages over HES 70.000. In some aspects, HES 130.000 seems preferable. To elucidate these differences in terms of clinical relevance, clinical validation is necessary.
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© 2000 International Anesthesia Research Society
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