Abstract
Background
Hydroxyethyl starch (HES) solutions are widely used for intravascular volume expansion. In Taiwan, the medium molecular weight of HES 200/0.5 and HES 130/0.4 solutions are most commonly used. It has been demonstrated that HES may affect coagulation and platelet function significantly. However, the differential effects of each medium molecular weight HES on platelets remain poorly reported. Therefore, we studied the influence of the two HES solutions on platelet function in vitro by mixing whole blood with different proportions of HES 130 kD, HES 200 kD, and saline to determine the differences.
Methods
Human blood samples for platelet function analyzer (PFA), aggregometry and blood/HES mixed test were drawn from the antecubital vein and put into test tubes containing 3.2% trisodium citrate (blood:citrate, 9:1). The specimens were divided into four groups, designated as whole blood, 10%, 20%, and 30% dilution with normal saline (N/S), HES130 or HES200 solution. The platelet function of each sample was measured by both PFA and platelet aggregometry.
Results
The results showed that the PFA-100 closure times CEPI-CT and CADP-CT were significantly prolonged in the samples diluted with normal saline, HES130 and HES200 than in the controls. The ADP triggered whole blood aggregometry showed that attenuated impedance was observed in samples of 20% diluted with HES130 and HES200 groups. The blood/HES mixed sedimentation test showed significantly increased proportion of the upper liquid layer in the HES200 group than in other groups.
Conclusion
Our data demonstrated that HES200 and HES130 possess noticeably inhibitory effects on platelet function, especially when the HES replaced proportion was more than 20%. HES200 has a greater effect on blood cells and plasma separation than does HES130.
Keywords
platelet aggregation; platelet function tests; starch, hydroxyethyl;
1. Introduction
Hydroxyethyl starch (HES) solutions have been widely used for volume expansion, maintaining hemodynamic stability and improving tissue perfusion.1,2 However, much controversy has arisen and continues with regard to the safety of various HES solutions3 because of possible side effects, such as antiplatelet function, increased bleeding tendency,4−9 renal function impairment and pruritus.10−12 These adverse effects seem to be closely related to the molecular weight and degree of substitution of blood by the HES solution. In general, the larger the molecular weight of the HES, the higher the frequency of adverse effects observed.13−17 However, this general rule does not correlate well with all clinical reports, in which the role of HES molecular weight attributive to adverse effects such as perioperative coagulopathy, blood loss, chest drainage and volume transfused remain an ongoing discussion.13,18−21
In Taiwan, HES solutions serve as the major volume expanders during anesthesia, and the medium molecular weight HES 200 kD/0.5 (10%)22 and HES 130 kD/0.4(6%)17,21 solutions are the most commonly used. However, the differential effects of the above mentioned medium molecular weight HES on coagulation and platelet function remain poorly reported.23 In addition, the controversial results documented in published clinical studies have aroused many criticisms for innate defects, such as lack of proper randomization, unreliable baseline laboratory data, different past history before study, and uncontrolled fluid administration during operation. Furthermore, the patients enrolled into the studies were of different pathophysiological status, which constituted a complex experimental system and tended to jeopardize the data consistency if the factors were not well-controlled. Therefore, we proposed to use a well-controlled ex vivo system to determine the effect of HES solution of different molecular weights on blood coagulation and platelet inhibition.
In the present study, we investigated the influence on coagulation and platelet function by incremental hemodilution of human blood from volunteers with either normal saline, HES130 or HES200. Blood coagulation was monitored by a platelet function analyzer (PFA-100; Dade Behring, Leiderbach, Germany) and platelet aggregation was examined by whole blood impedance aggregometry. In addition, the effect of HES solution on erythrocyte sedimentation rate (ESR) was measured as reference for dynamic viscosity and alteration of rheological properties during HES solution administration.
2. Materials and Methods
2.1. Blood sampling
Healthy volunteers with no history of hematological diseases, such as platelet or coagulation disorders, and taking neither aspirin nor other medication having an effect on coagulation function in the last 2 weeks, were recruited. All of the following experimental procedures and protocols were approved by the Institutional Review Board of Chang Gung Memorial Hospital, and all of the volunteers were subjected to venepuncture for 20 mL blood after their informed consent was obtained. Venous blood was sampled from the antecubital vein in the forearm using a polypropylene syringe with 20G needle. The sampled blood was then dispensed into polystyrene tubes containing 3.2% trisodium citrate (blood:citrate, 9:1) and stored at room temperature for 10 minutes. Platelet function was subsequently measured by the PFA-100 and platelet aggregometry (Chrono-log 560-CA; Chrono-Log Corp., State College, PA, USA). All measurements of samples were completed within 30 minutes to 2 hours of sample collection to ensure reliability. Samples were divided into 12 portions and categorized into four groups, designated as whole blood, 10%, 20%, and 30% normal saline (N/S) dilution HES130/0.4 (VoluVen® 6%; Fresenius Kabi Deutschland GmbH, Bad Homburg v.d.H., Germany) and HES200/0.5 (Haessteril® 10%; Fresenius Kabi Deutschland GmbH, Bad Homburg v.d.H., Germany).
2.2. PFA-100 studies
The PFA-100 (Dade Behring)24 evaluates platelet function by determining the time to occlusion of an aperture in membrane coated with collagen and adenosine diphosphate (ADP) or epinephrine (EPI) as citrated whole blood flows through under high shear stress conditions. The blood sample is aspirated under a constant negative pressure through a synthetic capillary with a coated membrane and passes through an aperture. In response to the stimulation by collagen and ADP present in the coating and the shear stresses at the aperture, platelets adhere and aggregate, and the platelet plug ultimately occludes the aperture. The time required to obtain full occlusion of the aperture is defined as the PFA-closure time. Measurements of time to aperture occlusion (closure time, CT) were performed using both collagen/EPI (CEPI) cartridge and collagen/ADP cartridge (CADP) (Dade Behring). After 300 seconds, the process is automatically terminated, inferring that CT is longer but will be reported as 300 seconds. According to the manufacturer, in healthy individuals, the normal range for CEPI closure time is 94−193 seconds, and for CADP closure time is 71−118 seconds. Citrated blood samples were first sat at room temperature for 10 minutes, and then a 1-mL aliquot of testing sample was made by mixing the sampled blood with normal saline, in the proportions of 1:9, 2:8 and 3:9 and HES130 and HES200 before analysis.
2.3. Impedance-based platelet aggregation studies
Blood samples were first collected into tubes containing 3.2% trisodium citrate and the whole blood aggregations were measured by changes in electrical impedance using a Chrono-log 560-CA electrical impedance aggregometer (Chrono-Log Corp.) as previously described.25 In brief, the control aliquots for testing were made by mixing 0.5 mL citrated-whole blood with 0.5 mL normal saline (37ºC). Then, the experimental aliquots for testing were made by diluting the whole blood samples with normal saline, in the proportions of 1:9, 2:8 and 3:9 and with HES130 and HES200. Each of 1000 μL testing samples in a polycarbonate cuvette (Chrono-Log Corp.) was placed in the aggregometer for constant shaking at a temperature of 37ºC with a stir bar set at a speed of 1000 rpm before testing. The electrodes were inserted into the cuvette and a current of small voltage was applied across the two wires; this constant impedance was measured as the baseline value, which is caused by the stable uniform platelet monolayer coating the two fine palladium wires on the electrode. As platelet aggregation is stimulated by either ADP (10 μM) or collagen (2 μg/mL) (Chrono-Log Corp.) after 4 minutes of incubation, the platelet coating on the palladium wires thicken over the next several minutes with a corresponding alteration in electrical impedance between the electrode wires. This change in impedance (indicated in ohms) is directly proportional to the extent of platelet aggregation. Whole blood aggregation is then expressed as the alteration in electrical impedance in ohms. Each of the aggregation curves were recorded for 6 minutes and analyzed by AGGROLINK software (Chrono-Log Corp.).
2.4. ESR measurement
The experimental samples were prepared as mentioned above, each of which was made into 1 mL aliquot by mixing whole blood with normal saline, in the proportions of 1:9, 2:8 and 3:7 and with HES130 or HES200. Thus, the samples in each parallel group came from the same donor in order to avoid individual bias. The samples were mixed by gentle up-and-down motion 10 times and then dispensed into a vertically placed 2-mL polycarbonate cuvette (Chrono-Log Corp.) for observation. The sedimentation of blood cells and the upper separating layer were measured at the 5th, 10th and 20th minutes. The classical Westergreen method was used to analyze the red blood cell (RBC) sedimentation rate as a measure of RBC aggregation. ESR was expressed as mm/hour.
2.5. Statistical analysis
All data were recorded and calculated using Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). Data were expressed as mean ± standard deviation and compared using nonparametric tests. The differences within each study group were evaluated by the t test. The significance of the differences between study groups was tested by Kruskal-Wallis oneway analysis of variance on ranks. A value of p< 0.05 was considered to be statistically significant.
3. Results
3.1. No significant difference was found between groups with same degree of hemodilution
The general appearances of data collected from complete blood count analysis of hemodilution made by different proportions of normal saline, HES130, and HES200 are shown in Table 1. As expected, both hematocrit and platelet count decreased decrementally from normal control value (42.27 ± 3.61% and 273.1 ± 47.0 × 109 /L, respectively) to the lowest value of 30% dilution (27.67 ± 2.79% and 189.3 ± 24.0 × 109 /L, respectively). There were no significant differences in hematocrit or in platelet count analysis among the normal saline, HES130, and HES200 groups for the same degree of dilution, which indicates a reliable basis of diluted blood samples for the subsequent studies.
3.2. Closure times of PFA-100 analysis were all prolonged in each group compared with undiluted whole blood samples
We first examined whether or not PFA-100 could sensitively detect the influence of coagulation by hemodilution. It was not surprising that, as demonstrated in Figure 1, significantly prolonged closure times collected from epinephrine and ADP cartridges testing were observed in all the diluted sample groups, regardless of the diluents or degrees of dilution. Thus, hemodilution-derived coagulopathy could be effectively validated by the prolonged closure times of PFA-100 analysis.
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3.3. Aspirin-like effects of both HES suggested from CEPI-CT experiments
As demonstrated in Figure 1A, our data further demonstrated significantly prolonged CEPI-CT in both HES groups, while in the dilution groups it was ≥ 20%. No significant difference in CEPI-CT was discovered among groups with 10% dilution. These data indicate that both HES130 and HES200 would exaggerate the hemodilution-derived coagulopathy, suggesting that HES per se, no matter of whatever molecular weight, would promote aspirin-like coagulation deficiency in a dose-dependent manner.
3.4. Antiplatelet and aspirin-like effects of HES suggested from CADP-CT analysis
In order to further elucidate the observed aspirinlike properties of two different HES in CEPI-CT experiments, we evaluated the CADP-CT to distinguish the direct antiplatelet effect from the aspirin-like effect. Interestingly, no significant difference in CADP-CT was observed among the groups < 20% dilution; however, HES200 resulted in longer CADPCT than did normal saline and HES130. These data reveal that the aspirin-like effect of the two HES is prominent when blood samples are diluted within 10−20%. In addition, the antiplatelet effect of HES200 was masked at 10−20% dilution but observed in 30% dilution, and the antiplatelet effect of HES130 could not be observed in PFA-100 closure time.
3.5. Antiplatelet effects of two different HES confirmed by ADP-dependent whole blood impedance aggregation analysis
For the purpose of answering the question of whether two different HES obtained antiplatelet function, we deployed ex vivo aggregation analysis by whole blood impedance aggregometry to highlight this issue. By using ADP as an inducer of aggregation, at 20% dilution, antiplatelet effect was masked in PFA-100 studies. But our data showed that aggregation impedance was significantly reduced in both HES130 (1.83 ± 2.35 ohms) and HES200 (1.51 ± 1.45 ohms) groups than in the control (10.86 ± 3.98 ohms) or normal saline (5.97 ± 4.61 ohms) groups (Figures 2A and 2C). However, this antiplatelet effect could not be observed significantly in collagendependent aggregation (Figures 2B and 2D). These results revealed that the specificity of HES mediated antiplatelet function and excluded the possibility of nonspecific platelet function deficiency oriented from hemodilution, indicating that HES might affect ADP-related receptor signaling pathways, such as P2Y1 and P2Y12.
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3.6. Acceleration in ESR observed most prominently in HES200 groups
To explore the possible alteration of blood rheological properties by HES in various flow situations, we probed the fundamental ex vivo behavior of RBCs by measuring the ESR. Our data demonstrated that both HES130 at 30% dilution and HES200 at > 20% dilution significantly accelerated ESR compared with the normal saline groups when measured at 20 minutes (Figure 3). Similar results were also observed at 5 and 10 minutes (data not shown). These results indicate that both HES130 and HES200 could possibly change the rheological properties of RBCs at 30% dilution. However, this ESR alteration was predominantly observed in HES200 groups even at 20% dilution.
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4. Discussion
In the present study, we demonstrated that both HES130 and HES200 interfered with blood coagulation and platelet function when compared with normal saline in ex vivo human blood studies detected by whole blood impedance aggregometry and PFA-100. Although attribution of prolonged coagulation time to hemodilution might be possible, it could be improved (but not totally restored) by fibrinogen supplementation, administration of washed platelet or factor VIII in spite of insignificant beneficial effect.20 The observed coagulopathy in our experimental setting could not be accounted for explicitly by hemodilution, since the inhibitory effects of platelet aggregation by both HES130 and HES200 was much more prominent than normal saline in ADP, suggesting that there is a direct negative effect of both HES130 and HES200 on platelet function. Although PFA-100 failed to differentiate the minor discrepancy of HES130 and HES200 in prolonging closure time, the data collected from impedance aggregometry indicated that HES200 slightly outweighed HES130 in terms of inhibitory effect on ADP-induced aggregation. Our results also showed that ESR was significantly increased by HES200 but not by HES130 or normal saline within 10−20% dilution, indicating that HES200 might alter blood and erythrocyte rheological properties under various flow conditions in the human body and could more easily cause the documented adverse effects such as renal function impairment.
The observed antiplatelet effect of HES130 and HES200 in our ex vivo studies were also found in previous reports, in which the mechanisms of HES that mediated antiplatelet effect were extensively studied.13,22,26−28 It was indicated that HES-related hemodilution per se would not contribute to its antiplatelet effect but could decrease the plasma concentration of von Willebrand factor and coagulation factor VIII.23,29 Interestingly, these coagulation deficiencies caused by hemodilution could not be totally restored by supplementation of both von Willebrand factor and coagulation factor VIII.20 It seems that HES will neither affect intracellular signaling of platelet activation nor intracellular calcium release.27 Data collected from flow cytometry revealed that HES suppresses the expression of GPIIb-IIIa on the platelet surface without affecting the expression of P-selectin and GPIb,22 principally by extracellular binding of HES to GPIIb-IIIa and subsequently blocking access to the platelet fibrinogen receptor.28 These results provide a plausible explanation for the observed suppression of ADPinduced platelet aggregation in our ex vivo experiments, whereas collagen-induced signaling is much more complicated than ADP and thus it might still keep its near maximum activation under the influence of HES. Therefore, solely decreasing GPIIb-IIIa by HES would not significantly affect collageninduced aggregation.
In this study, PFA-100 failed to distinguish the small discrepancy between HES130 and HES200, which is in sharp contrast to previous reports that HES200 had longer closure time than HES130.13,19 On the other hand, our observed indistinguishable whole blood coagulopathy is in line with other reports.20,21 Thus, whether HES130 has better hemostasis effect than HES200 remains controversial. This debatable effect on hemostasis between HES130 and HES200 extends from in vitro to in vivo studies. For example, recent publications demonstrated that less impairment of clot formation was noted in resuscitation of hemorrhagic shock with HES200 than with HES130.18 On the contrary, a slightly superior hypocoagulation effect with HES200 than with HES130 was observed in volume supplement studies during neuroanesthesia.30 Interestingly, the therapeutic equivalence of HES130 to HES200 in mean infused volume was also found in patients who received major gynecological surgery, regardless of reduced hematocrit in the HES200 group 6 hours after operation and lower international normalized ratio in patients who received HES130.31
Is the HES130 solution better than the standard HES200 in clinical practice? In terms of hemostasis, we cannot answer this question based on our ex vivo data since the slightly higher suppression of aggregation by HES200 could not be confirmed by PFA-100, although when compared with the control normal saline groups, both HES solutions prolonged PFA-100 closure time and significantly suppressed platelet aggregation. However, it has been suggested that HES solutions of lower molecular weight may degrade faster and have less influence on hemostasis than those of higher molecular weight.13,22,26−28 Moreover, there is accumulating evidence to support the clinical use of HES130. A convincing report based on retrograde analysis of pooled data from all available studies comparing HES130 and HES200 in major surgery, in which estimated blood loss, actual blood loss, transfused blood product volumes, drainage loss and coagulation variables were examined, revealed that significantly reduced blood loss and transfusion requirements were observed in patients who received HES130 during major operations.32 Therefore, HES130 rather than HES200 was recommended for use in clinical practice even though both are similar with regard to volume expansion efficacy.32
As we sought to determine whether HES of different molecular weights would alter ESR to represent a genuine influence on erythrocyte dynamic viscosity and rheological properties under various flow situations, we were surprised to discover that HES200 increased ESR significantly compared to HES130 and normal saline. Previous studies indicated that HES of different concentrations would first increase the viscosity of a 40% RBC suspension under both low and high shear stress and then increase RBC aggregation in ongoing phase. These studies reasonably support the concept that viscosity derived electrophoretic mobility of RBCs in HES is positively correlated with the concentration of HES, which is probably the result of an interaction of the electrostatic repulsive forces between the cells.33 However, whether or not the alteration in RBC dynamic viscosity and rheological properties can be linked with HES-related renal dysfunction is not known. To address this issue, a literature review was done. As documented, HES130 and HES200 impair renal function in major surgery34,35 or sepsis,36 which seems to be more prominent in patients with prior renal impairment.37 In contrast, a multicenter randomized study failed to show the correlation between acute kidney injury and HES administration in patients with sepsis38 and critical illness.39 These results are further supported by recent studies which are not in accord with the impact of prior renal dysfunction and choice of colloid for renal safety.40,41 Nonetheless, the general prevailing consensus is that HES130 has a faster clearance rate from the circulation than does HES200.42 Ongoing discussion with accumulating evidence also strongly argue about HES-related renal dysfunction. In general, it has been suggested that HES130 administration is associated with less renal dysfunction than HES200 and may safely be used perioperatively.43−45 Therefore, further efforts are needed to elucidate the relationship between observed ESR increase by HES200 and postoperative renal impairment.
In conclusion, our data collected from human ex vivo studies demonstrated that both HES200 and HES130 displayed prolongation of PFA-100 closure time and suppression of platelet aggregation. Additionally, HES200 promoted acceleration of ESR significantly compared with HES100 and normal saline. These results partly explain the phenomena observed in clinical studies, such as the association of different HES solutions and degree of blood loss. The ESR data also shed light on the possible causative relationship with renal dysfunction, which needs further investigations.
Acknowledgments
This work was supported by research grants from Chang Gung Memorial Hospital (CMRPG360331-2; CMRPG33015) and, in part, from the National Science Council (NSC 94-2314-B-182A-084). The authors would like to thank Miss Yi-Tsui Chu for her help in sample preparation and data analysis, and the kind volunteers who provided blood for the study.