Clinical indications for use of HES solutions include provision of rapid, sustained blood volume expansion during resuscitation as well as plasma oncotic support in conditions of hypoproteinemia. Administration of these synthetic colloids is not without risk, and reported adverse effects include allergenicity, kidney injury, accumulation in tissues, and a decrease in platelet function.1 Various formulations are available and are becoming more cost-effective for use in horses. Many reports2–9 describe the hemodynamic effects of HES solutions in horses; however, information regarding their oncotic effects and adverse effects is limited.
The adverse effects of colloids on platelet function and coagulation cannot be explained by dilution alone.1,10,11 Changes in coagulation following administration of HES solutions reportedly occur in humans,10,12,13 dogs,14,15 and horses.2,6,8 Because the adverse effects are dose related, recommendations have been made for maximum daily doses of HES products.10
Multiple mechanisms are responsible for coagulopathy induced through administration of HES solution. Interference with clotting factor function can happen when HES binds to circulating von Willebrand factor–factor VIII complexes and results in more rapid than usual removal of these complexes.10 Because these complexes allow platelet binding to sites of vascular injury, the increase in their removal results in platelet dysfunction.13 Incorporation of the colloid molecule into a clot results in a weaker clot and an increase in fibrinolysis.13 Administration of HES solutions also induces fibrinogen deficiency16 and further alters platelet function by modifying the platelet membrane.13
The effect of HES solutions on coagulation varies with the molecular composition of the product and carrier solution. Hydroxyethyl starch products with higher molecular weights and molar substitution ratios are more likely to interfere with coagulation than are products with a lower molecular weight and substitution ratio.1,10,11,13,17 Products in a balanced electrolyte solution might have less effect on coagulation than those in saline (0.9% NaCl) solution.14
In horses with colic requiring surgery, administration of a synthetic colloidal solution (10% pentastarch) can improve intraoperative cardiovascular indices to a greater degree and for a longer period than administration of an equal volume of hypertonic saline (7% NaCl) solution.3 In many of the aforementioned causes of hypovolemia as well as in chronic diseases such as Lawsonia intracellularis infection, loss of protein due to increased vascular permeability or gastrointestinal mucosal barrier compromise results in low plasma oncotic pressure.6,18 Colloidal solutions can be used to restore and maintain COP in these horses.5 For example, an oncotic effect can persist as long as 24 hours after an 8 to 10 mL/kg bolus of HES solution (6% hetastarch) is administered to hypoproteinemic horses.5
In horses with hypovolemia or hypovolemic shock secondary to strangulating and inflammatory causes of colic, endotoxemia, or sepsis, colloidal solutions are an attractive option for resuscitation because administration of a relatively small volume results in an increase in intravascular volume that is maintained for a longer duration than when a similar volume of crystalloid solution is administered.19,20 The use of colloidal solutions promotes rapid reversal of hypovolemia, thus preventing further problems caused by a decrease in perfusion and lessening the delay in providing other interventions (ie, surgery).
Hydroxyethyl starch solutions are categorized by their mean molecular weight (in kDa) and molar substitution ratio. A higher molar substitution ratio results in slower degradation and indicates a greater number of hydroxyethyl substitutions per glucose unit.21 Three HES solutions are available in the United States. Two are hetastarch solutions (HES 600/0.75 in saline [0.9% NaCl] solution and HES 670/0.75 in lactated Ringer's solution), and one is a newer generation tetrastarch (HES 130/0.4 in saline solution). To choose the most appropriate HES solution for use in horses, any difference among products in their coagulation effects needs to be known. Use of a product with minimal effects on coagulation is particularly important because many horses that would benefit from IV colloidal fluid administration may also be at increased risk for thrombotic complications or coagulopathy or may require surgery. For example, horses with gastrointestinal disease22 and foals with sepsis23,24 have a high risk of coagulopathy prior to receiving colloidal solutions.
Studies2,6,8 of the effects of HES solutions on coagulation in horses have been limited in number and scope. Only solutions with high molecular weight and molar substitutions have been evaluated, and coagulation has only been evaluated through coagulation factor analysis and coagulation times. Many of the coagulation effects involve platelet dysfunction and alterations in clot strength, so alternative methods of coagulation assessment may provide more information. Use of techniques to assess whole blood viscoelastic coagulation, such as thromboelastography and dynamic viscoelastic coagulometry, provides more information on the entire coagulation process, including clot strength and fibrinolysis, than the other methods. Tests such as optical platelet aggregometry and automated platelet function analysis provide more specific information about the effects of HES solutions on platelet function. The purpose of the study reported here was to determine whether in vitro addition of HES solutions to equine blood would cause hypocoagulation and decreased platelet function beyond that explainable by dilution alone. Furthermore, we hypothesized that these changes would be dose dependent and related to the molecular weight of the HES and to the electrolyte composition and buffering capacity of the carrier solution.
Materials and Methods
Animals—Seven healthy adult university-owned horses (6 geldings and 1 mare) with a mean ± SD age of 13.4 ± 6 years were used in the study. Horses were deemed healthy on the basis of physical examination, serum biochemical analysis, CBC, and routine coagulation analysis (prothrombin time, activated partial thromboplastin time, and plasma fibrinogen concentration). Approval for sample collection was granted by the Institutional Animal Care and Use Committee of the University of Georgia.
Sample collection—Atraumatic jugular venipuncture was performed by 1 investigator who used a 19-gauge, 3/4-inch butterfly catheter. Blood samples were collected into 20-mL syringes that had been prefilled with 2 mL of 3.2% sodium citrate so that a final citrate-to-blood ratio of 1:9 was achieved. The citrate and blood were mixed by gentle inversion of the syringe 5 times after sample collection and 5 times prior to transfer of aliquots into 15-mL conical tubes that contained the test solutions. For logistic reasons, blood samples were collected from each horse twice within 6 hours of each other to allow for all solutions to be tested in a timely manner.
Blood sample treatment—Each blood sample was subjected to 6 treatments: no additive (control sample), saline (0.9% NaCl) solution, lactated Ringer's solution, 6% HES 600/0.75 in saline solution,a 6% HES 670/0.75 in lactated Ringer's solution,b and 6% HES 130/0.4 in saline solution.c Each treatment fluid was added to the blood samples to create 1:8 and 1:4 dilutions. The 1:8 dilution was intended to simulate the effect of a 10 mL/kg bolus given to a horse with an estimated blood volume of 80 mL/kg, whereas the 1:4 dilution simulated a 20 mL/kg bolus. For each horse, the order in which the dilutions and fluids were analyzed was assigned by simple random selection so that the order of the analyses varied between diluents and dilutions. All samples were mixed by gentle inversion 5 times after treatment and before selection for analysis. The platelet count of all samples was determined with an automated analyzer.d
Samples were analyzed by means of thromboelastography, dynamic viscoelastic coagulometry, platelet function analysis, and optical platelet aggregometry. The COP was measured by use of platelet-poor plasma and a colloid osmometer.e Aliquots of citrated platelet-poor plasma were also frozen and sent as a batch to the Cornell University Comparative Coagulation Laboratory for determination of factor VIII:C and von Willebrand factor concentrations.
Thromboelastography—Tissue factor-activated thromboelastographyf was performed following a previously described technique.25 Briefly, samples for thromboelastography were allowed to rest for 30 minutes at room temperature (24° to 26°C). Twenty microliters of 0.2M CaCl2 and 10 μL of 1:100 recombinant human tissue factorg in 4% bovine serum albumin and PBS solution were added to a thromboelastography reaction cup warmed to 37°C. Next, 330 μL of citrated whole blood was added to the cup, partially aspirated back into the pipette tip to mix the contents, and then replaced into the cup to start the test. Thromboelastography analytic softwareh was used to display and calculate R (reaction time), K (clot formation time), α-angle, and MA.
Dynamic viscoelastic coagulometry—After samples were rested for 30 minutes at room temperature, 20 μL of 0.2M CaCl2 was added to a warmed (37°C), glass bead–activated cuvette, followed by 340 μL of whole blood. A magnetic stir bar included in the cuvette mixed the sample prior to initiation of testing with a dual-channel dynamic viscoelastic coagulometer.i Specialized softwarej was used to display the results and calculate ACT, clot formation rate, and clot retraction value (ie, platelet function).
Platelet function analysis—Cartridges containing collagen and ADP were prewarmed to room temperature, and 800 μL of whole blood was added, followed by analysis with a platelet function analyzer.k Closure time was measured and recorded for each sample in duplicate.
Aggregometry—Platelet aggregometry was performed with PRP and platelet concentrate. Platelet-rich plasma was prepared by centrifugation of citrated blood at 850 × g for 10 minutes at room temperature. The PRP was then aspirated, and the platelet count was determined.d The PRP was allowed to rest for 30 minutes at room temperature before analysis. The cells and remaining plasma were centrifuged at 1,500 × g for another 10 minutes to yield platelet-poor plasma to serve as a zero reference point. For each sample, 2 aliquots of PRP were analyzed with an optical aggregometer.l One was activated with collagen (10 μg/mL), and the other was activated with 15μM ADP. For each sample, percentage aggregation and the slope of the percentage aggregation over time tracing were determined with specialized software.m
Platelet concentrate was made by centrifugation of 1 mL of PRP in a microcentrifuge tube at 3,000 × g for 10 seconds. The supernatant was gently removed via aspiration from the pelleted platelets, which were then resuspended in 500 μL of PRP. The platelet count of the platelet concentrate was determined, and optical aggregometry was performed by means of collagen activation.
Statistical analysis—Data were assessed for normality of distribution with the Kolmogorov-Smirnov method and statistically analyzed as appropriate for the distribution. For parametric data, differences in coagulation and platelet function values for treated blood samples from those of the control sample were evaluated through ANOVA for repeated measures followed by adjustment for multiple comparisons with the Holm-Sidak method. Nonparametric data were analyzed via Friedman repeated-measures ANOVA on ranks, with adjustments for multiple comparisons performed by means of the Tukey test. Values of P that were considered significant were determined by the results of multiple-comparison adjustments. Parametric data are reported as mean ± SD, and nonparametric data are reported as median (interquartile range).
Results
Colloid osmotic pressure was significantly (P < 0.001) greater than in the control samples for all equine blood samples treated with all types of HES solutions (Table 1). No significant differences in platelet counts in whole blood or platelet concentrate were evident for the 1:8 dilution treatments (Table 2). At both the 1:8 and 1:4 dilutions, the PRP without additives had a significantly (P < 0.006) higher platelet count than any of the samples diluted with any solution. In the 1:4 dilution group, whole blood without additive had a significantly (P < 0.001 for all) higher platelet count than blood diluted with any solution. Of the platelet concentrate samples for the 1:4 dilution, only those from blood diluted with HES 600/0.75 in saline solution and HES 670/0.75 in lactated Ringer's solution had a significantly (P < 0.002 for both) lower platelet count than in samples with no additive.
Mean ± SD COP (mm Hg) in equine blood samples (n = 7/treatment) treated with various test solutions at 1:8 and 1:4 dilutions.
| Treatment | 1:8 | 1:4 |
|---|---|---|
| No treatment (control) | 16.8 ± 0.9*† | 16.6 ± 0.8 |
| Saline solution | 13.1 ± 0.6‡ | 10.5 ± 0.6‡ |
| Lactated Ringer's solution | 13.3 ± 0.7‡ | 10.6 ± 0.5‡ |
| HES 600/0.75 in saline solution | 17.9 ± 0.9*†‡ | 18.7 ± 0.8†‡ |
| HES 670/0.75 in lactated Ringer's solution | 17.8 ± 1.0*†‡ | 18.9 ± 0.8*†‡ |
| HES 130/0.4 in saline solution | 19.9 ± 1.0*†‡ | 21.4 ± 1.0*†‡ |
Values expressed as mean ± SD
Value is significantly (P < 0.001) different from the corresponding value for saline (0.9% NaCl) solution.
Value is significantly (P < 0.001) different from the corresponding value for lactated Ringer's solution.
Value is significantly (P < 0.001) different from the corresponding control value.
The institutional reference interval for COP is 19 to 24 mm Hg.
Mean ± SD platelet counts (× 103 platelets/μL) for equine whole blood samples (n = 7/treatment) and the platelet rich products created after sample treatment with various test solutions at 1:8 and 1:4 dilutions.
| 1:8 | 1:4 | |||||
|---|---|---|---|---|---|---|
| Treatment | Whole blood | PRP | Platelet concentrate | Whole blood | PRP | Platelet concentrate |
| No treatment (control) | 129.2 ± 24.7 | 122.6 ± 29.9 | 184.3 ± 53.8 | 133.5 ± 24.5 | 121.6 ± 25.0 | 231.2 ± 62.1 |
| Saline solution | 118.8 ± 21.4 | 110.6 ± 21.8* | 195.3 ± 47.8 | 115.7 ± 22.0* | 101.9 ± 15.8* | 183.2 ± 76.2 |
| Lactated Ringer's solution | 123.2 ± 17.4 | 112.3 ± 24.0* | 180.0 ± 45.3 | 109.5 ± 20.1* | 101.4 ± 22.2* | 191.4 ± 35.4 |
| HES 600/0.75 in saline solution | 111.3 ± 21.0 | 102.1 ± 23.2* | 162.7 ± 53.7 | 103.3 ± 26.0* | 89.9 ± 24.5* | 149.2 ± 31.2* |
| HES 670/0.75 in lactated Ringer's solution | 121.7 ± 29.9 | 107.1 ± 26.5* | 166.7 ± 60.0 | 100.0 ± 20.6* | 96.7 ± 22.2* | 133.4 ± 22.7* |
| HES 130/0.4 in saline solution | 117.0 ± 22.3 | 105.3 ± 25.5* | 179.8 ± 46.9 | 103.0 ± 19.5* | 99.6 ± 19.3* | 160.6 ± 18.3 |
Value is significantly (P < 0.006) different from the corresponding control value.
The institutional reference interval for equine platelet count is 98 × 103 platelets/μL to 210 × 103 platelets/μL.
Platelet function analysis revealed that closure time at the 1:8 dilution was significantly (P < 0.05) prolonged for blood samples treated with HES 600/0.75 in saline solution, compared with that for control samples (Table 3). At the 1:4 dilution, significant prolongations in closure time were detected for treatment with HES 600/0.75 in saline solution, HES 670/0.75 in lactated Ringer's solution, and HES 130/0.4 in saline solution, compared with closure time for the control samples.
Mean ± SD or median (interquartile range) platelet function closure times (seconds) for equine whole blood samples (n = 7/treatment) treated with various test solutions at 1:8 and 1:4 dilutions.
| Treatment | 1:8 | 1:4 |
|---|---|---|
| No treatment (control) | 80.7 ± 10.4 | 83.0 (78.8–90.0) |
| Saline solution | 93.4 ± 15.8 | 101.0 (88.5–107.6) |
| Lactated Ringer's solution | 88.1 ± 6.6 | 94.5 (87.6–97.3) |
| HES 600/0.75 in saline solution | 109.1 ± 24.5* | 117.0 (116.1–120.5)* |
| HES 670/0.75 in lactated Ringer's | 101.6 ± 26.6 | 109.0 (10.43–153.4)* |
| HES 130/0.4 in saline solution | 98.7 ± 10.2 | 119.5 (102.9–123.1)* |
Value is significantly (P < 0.05) different from the corresponding control value.
The institutional reference interval for closure time for equine whole blood is 75 to 102 seconds.
Thromboelastography parameters R, K, and α-angle were not significantly affected by dilution of blood samples with any of the HES solutions. Maximum amplitude was not significantly affected by the HES solutions at the 1:8 dilution (P = 0.947). However, at the 1:4 dilution, a significant (P < 0.001) decrease in MA was evident for blood sample treatment with HES 600/0.75 in saline solution (mean ± SD, 43.2 ± 7 mm), relative to the value for untreated samples (53.2 ± 5 mm).
Dynamic viscoelastic coagulometer ACT values were not significantly different at the 1:4 or 1:8 dilution of any of the HES solutions after correction for multiple comparisons; (Table 4). Clot formation rate was significantly (P < 0.003) lower for blood samples treated with 1:8 dilutions of HES 600/0.75 in saline solution and HES 670/0.75 in lactated Ringer's solution than for those treated with lactated Ringer's solution or saline solution alone. Clot formation rate was significantly (P < 0.005) lower in samples treated with 1:4 dilutions of HES 600/0.75 in saline solution, HES 670/0.75 in lactated Ringer's solution, and HES 130/0.4 in saline solution than in untreated samples and those treated with lactated Ringer's solution or saline solution alone. Platelet function was significantly (P < 0.007) decreased at both dilutions of HES 600/0.75 in saline solution, HES 670/0.75 in lactated Ringer's solution, and HES 130/0.4 in saline solution, compared with results for the control or diluent-alone treatments.
Mean ± SD values for results of dynamic viscoelastic coagulometry for equine whole blood samples (n = 7/treatment) treated with various test solutions at 1:8 and 1:4 dilutions.
| 1:8 | 1:4 | |||||
|---|---|---|---|---|---|---|
| Treatment | ACT (s) | Clot formation rate (Δ signal/s) | Platelet function (no units) | ACT (s) | Clot formation rate (Δ signal/s) | Platelet function (no units) |
| No treatment (control) | 336.6 ± 80.5 | 6.5 ± 2.5 | 2.9 ± 0.8 | 253.9 ± 45.9 | 11.3 ± 5.2 | 2.8 ± 0.8 |
| Saline solution | 371.1 ± 95.5 | 9.1 ± 3.2 | 2.9 ± 1.1 | 356.9 ± 52.2 | 9.9 ± 4.0 | 2.9 ± 1.1 |
| Lactated Ringer's solution | 379.4 ± 109.0 | 9.2 ± 3.1 | 2.9 ± 1.3 | 376.9 ± 58.9 | 11.0 ± 3.5 | 2.9 ± 1.3 |
| HES 600/0.75 in saline solution | 248.8 ± 75.6 | 3.5 ± 1.2* | 1.3 ± 0.4*† | 310.6 ± 122.9 | 4.4 ± 2.5*† | 1.3 ± 0.6*† |
| HES 670/0.75 in lactated Ringer's solution | 270.3 ± 110.4 | 3.9 ± 1.1* | 1.4 ± 0.9*† | 229.3 ± 88.1 | 2.5 ± 1.9*† | 0.8 ± 0.8*† |
| HES 130/0.4 in saline solution | 287.7 ± 51.3 | 6.0 ± 3.3 | 1.2 ± 0.5*† | 351.4 ± 95.3 | 4.2 ± 0.8*† | 1.2 ± 0.4*† |
Value is significantly (P < 0.003) different from values for saline solution and lactated Ringer's solution.
Value is significantly (P < 0.007) different from the corresponding control value.
The institutional reference intervals for equine blood are as follows: ACT, 212 to 330 seconds; clot formation rate, 6 to 26 Δ signal/s; and platelet function, 1.9 to 5.5 (no units).
Platelet aggregation after ADP stimulation did not differ in amplitude (P = 0.608) or slope (P = 0.093) for any treatments at the 1:8 dilution (Table 5). At the 1:4 dilution, all samples treated with an HES solution had a significantly lower (P < 0.005) amount of aggregation than did samples treated with only lactated Ringer's solution. Sample treatment with HES 600/0.75 in saline solution also resulted in a decreased amount of aggregation relative to sample treatment with saline solution alone (P = 0.004). The aggregation slopes for the 1:4 dilution of HES 600/0.75 in saline solution and HES 670/0.75 in lactated Ringer's solution were significantly (P < 0.003) lower than for that for lactated Ringer's solution alone. The slope for HES 600/0.75 in saline solution was also significantly (P = 0.004) lower than that for no treatment.
Mean ± SD percentage platelet aggregation with use of the agonist ADP (15μM) and slope (percentage aggregation/time) for PRP made from equine whole blood samples (7/treatment) treated with various test solutions at 1:8 and 1:4 dilutions.
| 1:4 | ||||
|---|---|---|---|---|
| Treatment | Aggregation (%) | Slope | Aggregation (%) | Slope |
| No treatment (control) | 49.4 ± 8.8 | 38.3 ± 9.2 | 48.8 ± 10.7 | 39.7 ± 6.1 |
| Saline solution | 49.0 ± 12.3 | 37.7 ± 7.3 | 48.1 ± 10.1 | 35.1 ± 9.2 |
| Lactated Ringer's solution | 46.3 ± 5.5 | 37.4 ± 10.0 | 53.3 ± 9.3 | 40.3 ± 11.7 |
| HES 600/0.75 in saline solution | 44.4 ± 12.1 | 33.6 ± 6.3 | 38.3 ± 8.9*† | 29.5 ± 9.1*‡ |
| HES 670/0.75 in lactated Ringer's solution | 47.3 ± 11.4 | 35.7 ± 10.7 | 38.4 ± 13.6* | 30.7 ± 7.5* |
| HES 130/0.4 in saline solution | 45.0 ± 7.6 | 31.7 ± 8.0 | 39.1 ± 10.6* | 32.1 ± 9.3 |
Value is significantly (P < 0.005) different from the corresponding lactated Ringer's solution value.
Value is significantly (P < 0.005) different from the corresponding saline solution value.
Value is significantly (P < 0.005) different from the corresponding control value.
In PRP treated with collagen to stimulate aggregation, significant decreases from the control sample existed for samples treated with HES 600/0.75 in saline solution at both dilutions (P < 0.001), HES 130/0.4 in saline solution at the 1:8 dilution (P = 0.003), and HES 670/0.75 in lactated Ringer's solution at the 1:4 dilution (P = 0.002; Table 6). No significant differences were evident in the slope of aggregation for any solution at either dilution. In platelet concentrate treated with collagen to stimulate aggregation, significant (P < 0.001) decreases from the control aggregation amplitude were evident for HES 600/0.75 in saline solution, HES 670/0.75 in lactated Ringer's solution, and HES 130/0.4 in saline solution at both dilutions. Additionally, a significant (P = 0.002) decrease in aggregation was detected between blood sample treatment with saline solution and treatment with HES 670/0.75 in lactated Ringer's solution at the 1:8 dilution. A significant (P < 0.004) decrease in aggregation was also found between treatment with lactated Ringer's solution and with the 1:4 dilutions of HES 600/0.75 in saline solution and HES 670/0.75 in lactated Ringer's solution.
Mean ± SD percentage platelet aggregation with use of the agonist collagen (10 μg/mL) and slope (percentage aggregation/time) for PRP (7/treatment) and platelet concentrate made from equine whole blood samples (7/treatment) treated with various test solutions at 1:8 and 1:4 dilutions.
| 1:8 PRP | 1:4 PRP | 1:8 platelet concentrate | 1:4 platelet concentrate | |||||
|---|---|---|---|---|---|---|---|---|
| Treatment | Aggregation (%) | Slope | Aggregation (%) | Slope | Aggregation (%) | Slope | Aggregation (%) | Slope |
| No treatment (control) | 76.9 ± 11.4 | 86.6 ± 10.1 | 72.3 ± 7.2 | 81.2 ± 22.6 | 72.2 ± 10.3 | 72.0 ± 10.7 | 69.4 ± 7.0 | 66.6 ± 4.6 |
| Saline solution | 73.5 ± 10.7 | 72.8 ± 12.2 | 61.6 ± 10.2 | 68.9 ± 18.8 | 65.8 ± 6.0 | 76.6 ± 14.7 | 56.0 ± 4.2 | 57.5 ± 5.5 |
| Lactated Ringer's solution | 71.0 ± 3.4 | 69.3 ± 9.5 | 68.7 ± 6.6 | 76.0 ± 9.0 | 59.5 ± 6.3* | 59.2 ± 9.4 | 57.8 ± 7.7 | 65.4 ± 11.1 |
| HES 600/0.75 in saline solution | 61.0 ± 9.1* | 81.3 ± 13.9 | 54.6 ± 9.5*‡ | 60.2 ± 5.8 | 56.8 ± 9.9* | 66.8 ± 8.3 | 43.4 ± 14.6*‡ | 53.5 ± 25.7 |
| HES 670/0.75 in lactated Ringer's solution | 65.8 ± 9.5 | 73.0 ± 13.5 | 58.5 ± 4.4* | 74.0 ± 15.1 | 49.7 ± 8.7*† | 63.7 ± 12.5 | 42.7 ± 11.6*‡ | 55.7 ± 19.9 |
| HES 130/0.4 in saline solution | 63.7 ± 10.4* | 69.6 ± 8.1 | 66.2 ± 2.6 | 74.9 ± 22.3 | 54.7 ± 10.3* | 59.2 ± 12.8 | 49.3 ± 7.9* | 63.2 ± 12.8 |
Value is significantly (P < 0.005) different from the corresponding control value.
Value is significantly (P < 0.005) different from the corresponding saline solution value.
Value is significantly (P < 0.005) different from the corresponding lactated Ringer's solution value.
A significant (P < 0.01) decrease in plasma concentration of von Willebrand factor: Ag, compared with the concentration in control samples, was detected for the 1:8 dilution of blood samples with HES 600/0.75 in saline solution and for all samples at the 1:4 dilution (Table 7). The percent activity of factor VIII:C was lower than control values for blood samples treated with saline solution, HES 600/0.75 in saline solution, and HES 130/0.4 in saline solution at the 1:8 dilution (P < 0.01) and for all treatments at the 1:4 dilution (P < 0.01).
Mean ± SD von Willebrand factor: Ag and factor VIII:C concentrations in equine blood samples (7/treatment) treated in vitro with various test solutions at 1:8 or 1:4 dilutions.
| 1:8 | 1:4 | |||
|---|---|---|---|---|
| Treatment | von Willebrand factor: Ag (%) | Factor VIII:C (%) | von Willebrand factor: Ag (%) | Factor VIII:C (%) |
| No treatment (control) | 94.6 ± 13.8 | 109.6 ± 27.5 | NM | NM |
| Saline solution | 87.4 ± 10.8 | 88.7 ± 23.4* | 76.1 ± 8.9* | 79.4 ± 18.2* |
| Lactated Ringer's solution | 87.0 ± 10.6 | 90.6 ± 21.0 | 77.7 ± 8.2* | 74.6 ± 16.3* |
| HES 600/0.75 in saline solution | 79.1 ± 20.4* | 86.9 ± 28.9* | 67.7 ± 16.4* | 70.6 ± 19.0* |
| HES 670/0.75 in lactated Ringer's solution | 90.3 ± 20.5 | 93.6 ± 14.6 | 69.0 ± 15.2* | 82.3 ± 19.4* |
| HES 130/0.4 in saline solution | 83.6 ± 18.5 | 88.1 ± 22.6* | 74.6 ± 16.3* | 80.4 ± 23.5* |
Value is significantly (P < 0.01) different from the corresponding control value.
NM = Not measured.
The institutional reference intervals for von Willebrand factor: Ag in horses is 65% to 165%, and for factor VIII:C is 50% to 200%.
Discussion
In the present study, 2 dilutions of various HES solutions and their carrier solutions were evaluated to determine the effect of HES solutions on coagulation and platelet function in equine blood samples in vitro. All dilutions and concentrations of the HES solutions evaluated resulted in an increase in COP when added to equine whole blood. The addition of all HES solutions also caused a prolongation in platelet function closure time at the 1:4 dilution, which was not apparent with the corresponding carrier solutions alone, despite equivalent volume-to-volume dilution. Prolongation of the closure time was also evident for blood sample treatment with HES 600/0.75 in saline solution at the 1:8 dilution. Given that a change in closure time was not detected with the other HES compounds at the 1:8 dilution, it may be that treatment with HES 600/0.75 in saline solution affects platelet function at lower doses than the other colloid products tested.
Numerous changes in values of coagulation parameters were revealed through dynamic viscoelastic coagulometry. At the lower (1:8) dilution, the clot formation rate was significantly lower for blood sample treatment with HES 600/0.75 in saline solution and HES 670/0.75 in lactated Ringer's solution, compared with the rate for their respective diluent solutions (saline solution and lactated Ringer's solution alone). This difference was not evident for treatment with HES 130/0.4 in saline solution. However, at 1:4 dilutions, all HES treatments caused a decrease in clot formation rate, compared with the rate for the carrier solutions and for no treatment. The clot formation rate represents the rate of fibrin formation following initiation of coagulation.26 Findings showed that not only were the HES molecules, rather than the carrier solutions, responsible for the changes observed, but the hetastarch solutions (HES 600/0.75 in saline solution and HES 670/0.75 in lactated Ringer's solution) also impacted the clot or fibrin formation rate, at a lower dilution than for the tetrastarch solution (HES 130/0.4 in saline solution). Treatments with all HES solutions at all dilutions resulted in a significant decrease in platelet function, which was not seen with the carrier solutions alone. The platelet function value is used to assess platelet function as measured by the timing and degree of clot retraction26 and likely indicates impairment of platelet function secondary to the HES solutions.
Overall, thromboelastography was not useful for detection of significant changes in coagulation caused by the addition of HES solutions to the equine whole blood samples. The 1 exception was HES 600/0.75 in saline solution at the 1:4 dilution, which yielded a lower MA than in the control samples. Considering that blood sample treatment with no other carrier or colloid solution resulted in a decrease in the MA, one might infer that the HES 600/0.75 molecule itself was responsible for the decrease in clot strength. The clot strength represented by the MA is dependent on platelet concentration, platelet function, and platelet-fibrin interaction.27 This thromboelastographic finding supports the platelet function finding that HES 600/0.75 in saline solution may decrease platelet function to a greater extent than the other colloids tested.
One possible reason for the difference between results of the 2 viscoelastic coagulation tests may lie in the method of clot activation. Whereas thromboelastography involved use of a strong activator of the extrinsic coagulation pathway (tissue factor), the dynamic viscoelastic coagulometer was activated with a strong activator of the intrinsic pathway. If most coagulation effects of the tested colloids result in impairment of the intrinsic pathway, then one might expect that a test of that pathway would be more sensitive to changes. Thromboelastography may also be activated with kaolin, which is an activator of the intrinsic pathway, and the possibility exists that this approach would increase the ability of thromboelastography to identify changes as a result of treatment with HES solution.
Optical aggregometry is a more specific method than thromboelastography for characterization of platelet-platelet interactions but involves use of PRP rather than whole blood. Various agonists can be used to initiate aggregation; however, equine platelets have been previously shown to be unresponsive to epinephrine and only have transient and reversible aggregation when exposed to serotonin and arachidonate.28 Collagen and ADP are the most commonly used agonists for evaluation of equine platelet aggregation. Adenosine diphosphate binds to specific receptors (P2Y1 and P2Y12), resulting in a change in platelet shape and release of contents of the α and dense granules, thereby causing platelet aggregation.29 Exposure of platelets to collagen (via glycoprotein VI and integrin α2β1) results in an influx of calcium, platelet shape change, and subsequent aggregation.30 Collagen-induced platelet activation is bolstered by thromboxane A2 production and platelet granule release, which provide a continued stimulus for aggregation (eg, via ADP- and 5-hydroxytryptamine–sensitive receptors).30
When ADP was used to initiate platelet aggregation in the present study, no changes in aggregation were detected at the 1:8 dilution of HES solutions; however, at the 1:4 dilution, changes were apparent in the maximal (percentage) aggregation and the slope, which represents the rate of aggregation. The percentage aggregation was lower in all HES-treated samples at the 1:4 dilution than in blood samples treated with lactated Ringer's solution, but no difference was identified between the HES-treated samples and the control. Blood samples treated with HES 600/0.75 in saline solution had a significantly lower degree of aggregation than did samples treated with saline solution alone. The slope for HES 600/0.75 in saline solution and HES 670/0.75 in lactated Ringer's solution was lower than that for lactated Ringer's solution as well. Given that blood sample treatment with the carrier solutions did not yield a lower degree of aggregation or slope relative to the control, the HES molecules and not their carrier solutions (or dilution alone) were the likely cause of the decreased platelet aggregation and the HES solutions likely had effects on platelet-platelet interactions. Some data regarding dogs suggest that the carrier solution of HES 670/0.75 in lactated Ringer's solution (which contains 4 mEq of calcium/L) may ameliorate some of the coagulation effects of the HES molecule.14 By direct comparison with the carrier solutions in the present study, the saline solution group did not result in decrements in platelet function, compared with the lactated Ringer's solution group, thus making it more likely that the observed changes were due to the HES. Although the HES solutions (HES 600/0.75 in saline solution and HES 670/0.75 in lactated Ringer's solution) affected both the slope and percentage aggregation, the tetrastarch HES 130/0.4 in saline solution only affected the percentage aggregation, suggesting that it may affect platelet function less than the 2 hetastarch solutions.
No changes in the slope of collagen-stimulated platelet aggregation were seen with either dilution of any solution, and these results were not different if PRP or platelet concentrate was studied. Decreased percentage aggregation was seen at the 1:8 dilution of PRP in both the HES 600/0.75 in saline solution and HES 130/0.4 in saline solution groups, whereas a decrease in aggregation was seen in the lactated Ringer's solution group and all HES groups with platelet concentrate. Similar results were achieved at the 1:4 dilutions, with a decrease in the degree of platelet aggregation in blood samples treated with HES 600/0.75 in saline solution and HES 670/0.75 in lactated Ringer's solution with PRP and in all HES-treated samples with platelet concentrate. Again, it did not appear that these findings were the result of dilutional effects. Although decreased aggregation was evident at both dilutions with HES 600/0.75 in saline solution, treatment with HES 130/0.4 in saline solution only caused a decrease at the 1:8 dilution and treatment with HES 670/0.75 in lactated Ringer's solution only caused a decrease at 1:4 dilution. Compared with the other 2 HES solutions, HES 600/0.75 in saline solution may have a greater propensity to decrease the degree of platelet aggregation.
Aggregometry was performed with PRP and platelet concentrate in an effort to correct for low platelet numbers in diluted PRP. Despite the fact that the platelet concentrate samples had the same or higher platelet count than that of the undiluted whole blood, more (rather than fewer) coagulation abnormalities were detected with the platelet concentrate. One possibility is that the additional manipulation of the platelets during preparation of platelet concentrate resulted in platelet activation or damage that decreased their aggregation responses. Because of the volume necessary to make platelet concentrate, only collagen was tested as an agonist; had ADP also been evaluated, conclusions might have been possible regarding specific versus global platelet dysfunction.
The results of plasma von Willebrand factor and factor VIII:C quantification appear to have been mostly attributable to hemodilution. The plasma concentration of von Willebrand factor was lower than the control sample only in the samples treated with HES 600/0.75 in saline solution at the 1:8 dilution, whereas the factor VIII:C concentration was lower than the control sample in the samples treated with saline solution for both HES 600/0.75 in saline solution and HES 130/0.4 in saline solution at the same dilution. At the 1:4 dilution, all test solutions resulted in a significant decrease in plasma von Willebrand factor and factor VIII:C concentrations, compared with control values, suggesting that hemodilution was responsible for the decrease at this dilution. One of the mechanisms believed to be related to HES-induced coagulopathy is an increase in the clearance from circulation of circulating von Willebrand factor–factor VIII complexes when they become bound by the HES.10 In vivo administration studies are necessary to determine whether clearance will result in a clinically relevant change in factor concentrations between the carrier and colloidal solutions.
Limitations of our study include the small number of horses from which blood samples were obtained and the in vitro nature of the test solution evaluations. As with any in vitro study, it is difficult to directly generalize the benchtop data to in vivo conditions. This may be particularly true in coagulation studies because coagulation does not rely on clotting factors and platelets alone but also on interactions with the endothelium and other cell types. The study was also performed in healthy adult horses. During inflammatory states, changes in coagulation and the endothelium can occur that may further alter the behavior of HES compounds in vivo.31
The platelet counts that were achieved in our PRP preparations were somewhat lower than expected, and this may have been the result of direct sample dilution as well as from activation and loss of platelets during PRP preparation. The method used for PRP preparation has been described,29,32–34 although the platelet count of the resultant PRP was not given in many of those descriptions. Other studies35–37 have involved use of various techniques to concentrate platelets obtained from PRP prior to aggregation. Platelet counts ranging from 100 × 103 platelets/μL to 300 × 103 platelets/μL do not affect the results of optical aggregometry34 and were difficult to avoid in the present study because of dilution. Future studies might be improved by adding a platelet concentration step prior to aggregometry.
In the present study, addition of HES solutions to blood samples from healthy adult horses caused changes in platelet function and the rate of fibrin formation, with a greater number of changes occurring at the higher (1:4) dilution. With the exception of the decrease in plasma von Willebrand factor and factor VIII:C concentrations at the higher dilution, these changes appeared to be attributable to the HES solutions, rather than the effects of dilution. In humans, greater coagulation changes occur with starches of higher versus lower molecular weight and a higher HES substitution ratio. On the basis of our data from dynamic viscoelastic coagulometry and platelet aggregometry, the same appears to be true for horses; the hetastarch solutions appeared to affect coagulation at a lower concentration than the tetrastarch HES 130/0.4 in saline solution.
Although the data did not suggest that treatment with hetastarch solutions impaired coagulation to a greater degree than did treatment with the tetrastarch, such a finding may be discovered through in vivo investigations or with a larger sample size, particularly given the different clearance characteristics between the hetastarch and tetrastarch solutions. Among the HES solutions, HES 600/0.75 in saline solution caused a greater number of significant coagulation disturbances when mixed with equine blood samples, compared with HES 670/0.75 in lactated Ringer's solution. These 2 starches are similar in molecular composition, with the only differences being that the mean molecular weight of HES 670/0.75 in lactated Ringer's solution is slightly higher than that of HES 600/0.75 in saline solution and that the carrier solutions are different. The carrier solutions alone appeared to result in few coagulation abnormalities, but it is possible that when combined with an HES, the carrier solution composition becomes more important in regard to coagulation effects. A study38 in humans demonstrated a much greater decrease in platelet aggregation response to ADP with HES 130/0.42 in saline solution than with a balanced electrolyte carrier. Findings of that study and the present study suggest that when combined with HES, the carrier solution becomes important in regard to coagulation effects. Alternatively, the difference in molecular composition may be enough to result in the different coagulation effects characteristic of these 2 solutions.
Use of HES solutions increases COP; however, these solutions also have dose-related impacts on coagulation, as evidenced by changes in platelet aggregation, clot rate and platelet function, and platelet function closure time. The clinical impacts of these in vitro coagulation changes in horses, particularly ill horses, have yet to be elucidated.
ABBREVIATIONS
| ACT | Activated coagulation time |
| COP | Colloid osmotic pressure |
| HES | Hydroxyethyl starch |
| MA | Maximum amplitude |
| PRP | Platelet-rich plasma |
Hespan, B Braun Medical Inc, Irvine, Calif.
Hextend, Hospira Inc, Lake Forest, Ill.
Voluven, Fresenius Kabi Austria, Graz, Austria.
CBC-diff, Heska Corp, Loveland, Colo.
4420 Colloid Osmometer, Wescor, Logan, Utah.
TEG 5000, Haemoscope, Braintree, Mass.
Innovin, Dade-Behring, Newark, Del.
TEG analytical software, version 4.2.95, Haemoscope, Niles, Ill.
Sonoclot, SCL-2, Sienco, Arvada, Colo.
Signature viewer software, Sienco, Arvada, Colo.
PFA-100, Siemens, Deerfield, Ill.
Platelet Aggregometer, Chrono-log, Havertown, Pa.
Aggro/Link, Chrono-log, Havertown, Pa.
References
1. Jungheinrich C. The starch family: are they all equal? Pharmacokinetics and pharmacodynamics of hydroxyethyl starches. Transfus Altern Transfus Med 2007; 9:152–163.
2. Jones PA, Tomasic M, Gentry PA. Oncotic, hemodilutional, and hemostatic effects of isotonic saline and hydroxyethyl starch solutions in clinically normal ponies. Am J Vet Res 1997; 58:541–548.
3. Hallowell GD, Corely KT. Preoperative administration of hydroxyethyl starch or hypertonic saline to horses with colic. J Vet Intern Med 2006; 20:980–986.
4. Pantaleon LG, Furr MO, McKenzie HC II, et al. Cardiovascular and pulmonary effects of hetastarch plus hypertonic saline solutions during experimental endoxtoxemia in anesthetized horses. J Vet Intern Med 2006; 20:1422–1428.
5. Jones PA, Bain FT, Byars TD, et al. Effects of hydroxyethyl starch infusion on colloid oncotic pressure in hypoproteinemic horses. J Am Vet Med Assoc 2001; 218:1130–1135.
6. Schusser GF, Reickhoff K, Ungemach FR, et al. Effect of hydroxyethyl starch solution in normal horses and horses with colic or acute colitis. J Vet Med A Physiol Pathol Clin Med 2007; 54:592–598.
7. Cohen ND, Woods AM. Characteristics and risk factors for failure of horses with acute diarrhea to survive: 122 cases (1990–1996). J Am Vet Med Assoc 1999; 214:382–390.
8. Pantaleon LG, Furr MO, McKenzie HC, et al. Effects of small- and large-volume resuscitation on coagulation and electrolytes during experimental endotoxemia in anesthetized horses. J Vet Intern Med 2007; 21:1374–1379.
9. Southwood LL, Dolente BA, Lindborg S, et al. Short-term outcome of equine emergency admissions at a university referral hospital. Equine Vet J 2009; 41:459–464.
10. Kozek-Langenecker SA. Effects of hydroxyethyl starch solutions on hemostasis. Anesthesiology 2005; 103:654–660.
11. Westphal M, James MF, Kozek-Langenecker S, et al. Hydroxyethyl starches: different products—different effects. Anesthesiology 2009; 111:187–202.
12. Martin G, Bennett-Guerrero E, Wakeling H, et al. A prospective, randomized comparison of thromboelastographic coagulation profile in patients receiving lactated Ringer's solution, 6% hetastarch in a balanced-saline vehicle, or 6% hetastarch in saline during major surgery. J Cardiothorac Vasc Anesth 2002; 16:441–446.
13. Van der Linden P, Ickx B. The effects of colloid solutions on hemostasis. Can J Anaesth 2006; 53:S30–S39.
14. Wierenga JR, Jandrey KE, Haskins SC, et al. In vitro comparison of the effects of two forms of hydroxyethyl starch solutions on platelet function in dogs. Am J Vet Res 2007; 68:605–609.
15. Smart L, Jandrey KE, Kass PH, et al. The effect of hetastarch (670/0.75) in vivo on platelet closure time in the dog. J Vet Emerg Crit Care (San Antonio) 2009; 19:444–449.
16. Fenger-Eriksen C, Tonnesen E, Ingerslev J, et al. Mechanisms of hydroxyethyl starch-induced dilutional coagulopathy. J Thromb Haemost 2009; 7:1099–1105.
17. Franz A, Braunlich P, Gamsjager T, et al. The effects of hydroxyethyl starches of varying molecular weights on platelet function. Anesth Analg 2001; 92:1402–1407.
18. Frazer ML. Lawsonia intracellularis infection in horses: 2005–2007. J Vet Intern Med 2008; 22:1243–1248.
19. Silverstein D, Aldrich J, Haskins S, et al. Assessment of changes in blood volume in response to resuscitative fluid administration in dogs. J Vet Emerg Crit Care 2005; 15:185–192.
20. Strunden MS, Heckel K, Goetz AE, et al. Perioperative fluid and volume management: physiological basis, tools, and strategies. Ann Intensive Care 2011; 1:2.
21. Jungheinrich C, Neff TA. Pharmacokinetics of hydroxyethyl starch. Clin Pharmacokinet 2005; 44:681–699.
22. Dolente BA, Wilkins PA, Boston RC. Clinicopathologic evidence of disseminated intravascular coagulation in horses with acute colitis. J Am Vet Med Assoc 2002; 220:1034–1038.
23. Bentz AI, Palmer JE, Dallap BL, et al. Prospective evaluation of coagulation in critically ill neonatal foals. J Vet Intern Med 2009; 23:161–167.
24. Dallap Schaer BL, Bentz AI, Boston RC, et al. Comparison of viscoelastic coagulation analysis and standard coagulation profiles in critically ill neonatal foals to outcome. J Vet Emerg Crit Care (San Antonio) 2009; 19:88–95.
25. Epstein KL, Brainard BM, Lopes M, et al. Thrombelastography in 26 healthy horses with and without activation by recombinant human tissue factor. J Vet Emerg Crit Care (San Antonio) 2009; 19:96–101.
26. Roberts DE, McNicol A, Bose R. Mechanism of collagen activation in human platelets. J Biol Chem 2004; 279:19421–19430.
27. Riewald M, Ruf W. Scientific review: role of coagulation protease cascades in sepsis. Crit Care 2003; 7:123–129.
28. Mateos-Trigos G, Evans R, Heath M. Effects of a P2Y(12) receptor antagonist on the response of equine platelets to ADP, comparison with human platelets. Res Vet Sci 2002; 73:171–175.
29. Ganter MT, Hofer CK. Coagulation monitoring: current techniques and clinical use of viscoelastic point-of-care coagulation devices. Anesth Analg 2008; 106:1366–1375.
30. Casella S, Giudice E, Giannetto C, et al. Effects of hydrocortisone and aminophylline on the aggregation of equine platelets. J Vet Sci 2011; 12:215–219.
31. Kingston JK, Bayly WM, Sellon DC, et al. Effects of sodium citrate, low molecular weight heparin, and prostaglandin E1 on aggregation, fibrinogen binding, and enumeration of equine platelets. Am J Vet Res 2001; 62:547–554.
32. Reikvam H, Steien E, Hauge B, et al. Thrombelastography. Transfus Apheresis Sci 2009; 40:119–123.
33. Myers KM, Lindner C, Grant B. Characterization of the equine platelet aggregation response. Am J Vet Res 1979; 40:260–264.
34. Brainard BM, Epstein KL, LoBato D, et al. Effects of clopidogrel and aspirin on platelet aggregation, thromboxane production, and serotonin secretion in horses. J Vet Intern Med 2011;25:116–122.
35. Brunso L, Segura D, Monreal L, et al. The secretory mechanisms in equine platelets are independent of cytoskeletal polymerization and occur through membrane fusion. Platelets 2010; 21:658–666.
36. Norris JW, Pratt SM, Auh JH, et al. Investigation of a novel, heritable bleeding diathesis of Thoroughbred horses and development of a screening assay. J Vet Intern Med 2006; 20:1450–1456.
37. Textor JA, Norris JW, Tablin F. Effects of preparation method, shear force, and exposure to collagen on release of growth factors from equine platelet-rich plasma. Am J Vet Res 2011; 72:271–278.
38. Schaden E, Wetzel L, Kozek-Langenecker S, et al. Effect of the carrier solution for hydroxyethyl starch on platelet aggregation and clot formation. Br J Anaesth 2012; 109:572–577.
