Original Study

Journal of Veterinary Emergency and Critical Care 25(4) 2015, pp 474–487 doi: 10.1111/vec.12320

Comparison of the in vitro effects of saline, hypertonic hydroxyethyl starch, hypertonic saline, and two forms of hydroxyethyl starch on whole blood coagulation and platelet function in dogs Virginie A. Wurlod, Dr. vet. med.; Judith Howard, Dr. vet. med., DACVIM; Thierry Francey, Dr. vet. med., DACVIM; Ariane Schweighauser, Dr. vet. med., DACVIM and Katja N. Adamik, Dr. vet. med., DACVECC Abstract

Objective – To compare the in vitro effects of hypertonic solutions and colloids to saline on coagulation in dogs. Design – In vitro experimental study. Setting – Veterinary teaching hospital. Animals – Twenty-one adult dogs. Interventions – Blood samples were diluted with saline, 7.2% hypertonic saline solution with 6% hydroxyethylstarch with an average molecular weight of 200 kDa and a molar substitution of 0.4 (HH), 7.2% hypertonic saline (HTS), hydroxyethyl starch (HES) 130/0.4 or hydroxyethyl starch 600/0.75 at ratios of 1:22 and 1:9, and with saline and HES at a ratio of 1:3. Measurements and Main Results – Whole blood coagulation was analyzed using rotational thromboelastometry (extrinsic thromboelastometry-cloting time (ExTEM-CT), maximal clot firmness (MCF) and clot formation time (CFT) and fibrinogen function TEM-CT (FibTEM-CT) and MCF) and platelet function was analyzed using a platelet function analyzer (closure time, CTPFA ). All parameters measured were impaired by saline dilution. The CTPFA was prolonged by 7.2% hypertonic saline solution with 6% hydroxyethylstarch with an average molecular weight of 200 kDa and a molar substitution of 0.4 (HH) and HTS but not by HES solutions. At clinical dilutions equivalent to those generally administered for shock (saline 1:3, HES 1:9, and hypertonic solutions 1:22), CTPFA was more prolonged by HH and HTS than other solutions but more by saline than HES. No difference was found between the HES solutions or the hypertonic solutions. ExTEM-CFT and MCF were impaired by HH and HTS but only mildly by HES solutions. At clinically relevant dilutions, no difference was found in ExTEM-CFT between HTS and saline or in ExTEM-MCF between HH and saline. No consistent difference was found between the 2 HES solutions but HH impaired ExTEM-CFT and MCF more than HTS. At high dilutions, FibTEM-CT and -MCF and ExTEM-CT were impaired by HES. Conclusions – Hypertonic solutions affect platelet function and whole blood coagulation to a greater extent than saline and HES. At clinically relevant dilutions, only CTPFA was markedly more affected by hypertonic solutions than by saline. At high dilutions, HES significantly affects coagulation but to no greater extent than saline at clinically relevant dilutions. (J Vet Emerg Crit Care 2015; 25(4): 474–487) doi: 10.1111/vec.12320 Keywords: canine, colloids, dilutional coagulopathy, platelet function analyzer, ROTEM, thromboelastometry From the Division of Small Animal Emergency and Critical Care, (Wurlod, Adamik) Small Animal Internal Medicine, (Francey, Schweighauser), and the Diagnostic Laboratory, (Howard) Department of Veterinary Clinical Medicine, Vetsuisse Faculty, University of Bern, Bern, Switzerland. Judith Howard contributed equally as first author and Katja N. Adamik is the senior author. The authors declare no conflict of interest. Address correspondence and reprint request to Dr. Virginie A. Wurlod, Small Animal Emergency and Critical Care Service, Veterinary Teaching Hospital, University of Illinois, IL, USA. Email: [email protected] Submitted June 26, 2013; Accepted November 22, 2013.

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Abbreviations

CFT CT CTPFA ExTEM FibTEM HES

clot formation time clotting time closure time extrinsic thromboelastometry fibrinogen function TEM hydroxyethyl starch

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Effects of volume expanders on hemostasis

HES 130/0.4

hydroxyethyl starch with an average MW of 130 kDa and a MS of 0.4 HES 600/0.75 hydroxyethyl starch with an average MW of 600 kDa and a MS of 0.75 HH 7.2% hypertonic saline solution with 6% hydroxyethylstarch with an average MW of 200 kDa and a MS of 0.4 HTS hypertonic saline solution MCF maximal clot firmness Ms molar substitution Mw molecular weight ROTEM rotational thromboelastometry

Introduction Hydroxyethyl starch (HES) solutions are artificial colloid solutions widely used in veterinary medicine for intravascular volume expansion. However, much controversy has arisen and continues in human medicine with regards to the safety of HES solutions because of their ability to impair coagulation. An increased risk of bleeding after infusion of HES is believed to be due not only to hemodilution but also to both direct and indirect effects of HES on components of the hemostatic system, including impaired platelet adhesion and aggregation, decreased coagulation factors, such as von Willebrand factor and factor VIII, decreased plasma fibrinogen concentrations, and enhanced fibrinolysis.1–5 Some studies have shown that HES solutions with a molecular weight (MW) above 200 or a molar substitution (MS) above 0.5, such as hydroxyethyl starch with an average MW of 600 kDa and a molar substitution of 0.75 (HES 600/0.75), have more pronounced effects on platelet function compared to solutions with lower MW or MS.6 The more recently introduced hydroxyethyl starch with an average MW of 130 kDa and a molar substitution of 0.4 (HES 130/0.4) is therefore claimed to have fewer adverse effects on hemostasis than previously used HES solutions.7 In addition, HES 130/0.4 is associated with shorter intravascular retention times and higher safety margins than HES with higher MW or MS.4,8 The concept of small volume resuscitation using hypertonic solutions involves the rapid infusion of a small dose (4 mL/kg body weight) of 7.2% hypertonic saline solution (HTS) or 7.2% HTS/colloid solution for primary resuscitation in trauma or shock patients.9–12 The rationale to this therapy is based on the instantaneous mobilization of endogenous fluids along osmotic gradients from the intracellular to the intravascular compartments, and is widely used in human medicine.9 In addition, resuscitation with hypertonic solutions has been extensively investigated in a variety of laboratory and clinical studies in dogs with hemorrhagic shock, and has been  C Veterinary Emergency and Critical Care Society 2015, doi: 10.1111/vec.12320

shown to have beneficial effects on circulatory parameters, microvascular perfusion, and oxygen delivery.11,13,14 Further studies have shown hypertonic solutions to be effective in resuscitation of endotoxic and septic shock, and shock associated with gastric dilation-volvulus, as well as in decreasing intracranial pressure when hemorrhagic shock was associated with intracranial hypertension in dogs.10,15–23 Synthetic colloids administered simultaneously with HTS increase intravascular volume for a longer period of time than HTS alone and exhibit an additive acute beneficial circulatory effect.9,24 However, besides the negative effects of HES on coagulation, HTS has also been shown to impair coagulation and platelet function in people.12,25 Only a few studies have evaluated the effects of HES or HTS on whole blood coagulation and platelet function in dogs.6,26–28,a The present study proposes an in vitro comparison of the effects of different IV fluid solutions (7.2% HTS, HH, HES 130/0.4 solution, and HES 600/0.75 solution) on whole blood coagulation and platelet function in canine blood using rotational thromboelastometry (ROTEM) and platelet function analysis. We compared the effects of fluids at both equal dilutions and at clinically relevant dilutions equivalent to approximately 33% blood dilution with saline (30 mL/kg), 11% blood dilution with colloids (10 mL/kg), and 4.4% blood dilution with hypertonic solutions (4 mL/kg). Our first hypothesis was that HH and 7.2% HTS cause greater impairment of whole blood coagulation and platelet function than HES solutions. Our second hypothesis was that HES 130/0.4 causes less in vitro impairment of whole blood coagulation and platelet function than HES 600/0.75.

Materials and Methods Animals Twenty-one healthy adult staff-owned dogs of both sexes with a minimum body weight of 20 kg were involved in the study. Dogs were determined to be healthy on the basis of history, physical examination findings, and results of a complete blood cell count (CBC) and serum biochemical analysis. Dogs were included if they had no history or evidence of recent illness or any chronic medical condition, and results of the CBC and serum biochemistry were within reference ranges. Dogs were excluded if nonsteroidal anti-inflammatory drugs, synthetic colloids, or anticoagulants had been administered within the previous 4 weeks. The study protocol was approved by the Animal Experiment Committee of the University of Bern. Rotational thromboelastometry Whole blood coagulation was assessed in citrated whole blood samples using a ROTEM 4-channel analyzer,b 475

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Table 1: Measurement of closure time (CTPFA , Platelet Function Analyzer 100), clotting time (CT, ROTEM), clot formation time (CFT, ROTEM), and maximal clot firmness (MCF, ROTEM) in undiluted canine whole blood samples and after dilutions with 0.9% saline solution (0.9% NaCl), 7.2% hypertonic saline solution (7.2% HTS), hypertonic hydroxyethyl starch (HH), hydroxyethyl starch 130/0.4 (HES 130/0.4), and hydroxyethyl starch 600/0.75 (HES 600/0.75)

PFA (n = 20)

ExTEM (n = 10)

FibTEM (n = 10)

Solution, dilution

CTPFA (s)

CT (s)

CFT (s)

MCF (mm)

CT (s)

MCF (mm)

Undiluted 0.9% NaCl, 1:22 0.9% NaCl, 1:9 0.9% NaCl, 1:3 7.2% HTS, 1:22 7.2% HTS, 1:9 HH, 1:22 HH, 1:9 HES 130/0.4, 1:22 HES 130/0.4, 1:9 HES 130/0.4, 1:3 HES 600/0.75, 1:22 HES 600/0.75, 1:9 HES 600/0.75, 1:3

63.0 ± 9.2 66.5 ± 10.2 70.8 ± 12.2 83.1 ± 10.6 111.2 ± 29.2∗ >300 100.9 ± 20.3∗ >300 66.6 ± 7.3 71.8 ± 7.1 87.8 ± 13.1 71.4 ± 9.6 66.3 ± 7.0 86.3 ± 5.9

32.0 (25–47) 32.0 (30–50) 45.5 (33–67) 42.5 (32–62) 33.0 (22–48) 37.0 (33–45) 38.5 (33–57)∗ 40.0 (35–48) 38.5 (23–58)∗ 45.5 (38–79) 104.0 (84–126)∗ 42.5 (30–70)∗ 38.0 (30–69) 64.5 (48–118)∗

101.0 (65–138) 117.0 (75–153) 124.0 (80–159) 161.0 (92–196) 154.0 (97–205)∗ 280.5 (150–364)∗ 186.0 (110–224)∗ 350.0 (199–441)∗ 129.0 (76–163) 144.5 (83–199)∗ 172.5 (90–255) 123.5 (73–154) 140.0 (94–164)∗ 197.5 (113–250)∗

63.0 (55–77) 60.5 (53–73) 56.5 (41–71) 53.5 (44–68) 56.0 (50–69)∗ 45.5 (40–61)∗ 52.5 (41–66)∗ 44.0 (37–60)∗ 59.5 (50–73) 57.0 (43–72) 48.0 (37–65)∗ 59.0 (51–73)∗ 57.0 (51–71) 50.0 (42–63)∗

32.5 (30–39) 31.0 (30–39) 37.5 (30–55) 36.5 (28–55) 33.0 (28–43) 40.0 (30–53) 35.0 (31–59) 39.5 (33–43) 37.5 (28–48)∗ 40.0 (35–46) 284.0 (87–1070)∗ 36.5 (35–43)∗ 45.5 (32–73) 93.5 (58–397)∗

9.5 (3–12) 8.5 (5–12) 7.5 (4–11) 5.0 (4–10) 8.0 (5–10) 7.0 (4–10) 7.0 (4–11) 5.0 (4–8)∗ 7.5 (4–10) 5 (3–7)∗ 3 (0–7)∗ 7.0 (5–9) 6.0 (4–8)∗ 3.0 (0–3)∗

Values are presented as mean ± SD for PFA-100 results and as median (range) for ROTEM ExTEM and FibTEM results. ∗ Values where a significant difference (P < 0.05) was found between saline and other solutions at the same dilution.

Figure 1: Solutions by dilution ExTEM CT: Comparison of in vitro effects on ROTEM ExTEM clotting time (ExTEM CT) of various solutions at different dilutions in canine blood. Results are displayed as median (bars) with 95% confidence intervals (error bars). The baseline measurement in whole blood is shown as a dashed line for reference. Significant differences between solutions at the same dilution are shown as a connector and asterisk.

which provides a continuous measure of clot firmness and global assessment of the hemostatic process. The method was recently described in detail elsewhere.29–31 Analyses performed included extrinsic thromboelastometry (ExTEM)c and FibTEM.d ExTEM measures clot formation triggered by the tissue factor dependent pathway 476

and gives rapid assessment of clot formation. The result is influenced by extrinsic coagulation factors, platelets, and fibrinogen. FibTEM is based on ExTEM for the fibrin part of the clot but contains cytochalasin D to inhibit the action of platelets and eliminates the contribution of platelets to clot formation. For ExTEM analysis, the  C Veterinary Emergency and Critical Care Society 2015, doi: 10.1111/vec.12320

Effects of volume expanders on hemostasis

Figure 2: Clinical dilutions ExTEM CT: Comparison of in vitro effects on ROTEM ExTEM clotting time (ExTEM CT) of various solutions in dilutions mimicking clinical doses in canine blood. Baseline measurements in whole blood are shown as a reference. Significant differences between solutions are shown as a connector and asterisk.

following measures were obtained: clotting time (CT) in seconds, clot formation time (CFT) in seconds, and maximal clot firmness (MCF) in millimeters. For FibTEM, only CT and MCF were recorded. The blood was maintained at room temperature (approximately 24°C) and all analyses were completed within 2 hours of blood collection32 and were performed according to the manufacturer’s instructions. Samples associated with error readings were discarded and measurements were repeated. Platelet function analysis Platelet function was assessed using a bench-top platelet function analyzer (PFA-100).e This point-of-care primary hemostasis analyzer, previously described elsewhere,33 assesses platelet function under conditions of shear stress and attempts to mimic conditions under which a platelet plug is formed in vivo,6 assessing both platelet adhesion and aggregation. The time in seconds needed for occlusion of the aperture by plug formation, called closure time (CTPFA ), is recorded in seconds from 40 to 300 s by the analyzer. For this study, collagen/ADP cartridgesf were used. All analyses were completed within 4 hours of blood sample collection. CTPFA recorded as >300 seconds by the PFA-100 analyzer were assigned a CTPFA of 300 s for statistical analyses. Procedure Twenty-five milliliters of venous blood were obtained by careful lateral saphenous venipuncture from each  C Veterinary Emergency and Critical Care Society 2015, doi: 10.1111/vec.12320

dog using a 20-Ga butterfly needle with an extension and adapter and distributed directly into the following blood tubes: EDTAg for CBC, lithium-heparinh for serum biochemical analyses, buffered 3.8% sodium-citratei for PFA-100 analyses, and 3.8% sodium-citratej for ROTEM analyses. Dogs were divided in 2 groups of 10 animals for ExTEM and FibTEM analyses, respectively, to enable all ROTEM measurements to be carried out within the allocated 2 hours of blood collection. Measurement of baseline CTPFA , CT, CFT, and MCF were performed in each dog. Blood samples were then divided into aliquots and diluted as follows: 0.9% saline solutionk (0.9% NaCl) in dilutions of 1:22 (ie, 1 part 0.9% NaCl and 22 parts blood), 1:9 and 1:3; 7.2% HTSl in dilutions of 1:22 and 1:9; HHm in dilutions of 1:22 and 1:9; 6% HES 130/0.4n in dilutions of 1:22, 1:9, and 1:3; and 6% HES 600/0.75o in dilutions of 1:22, 1:9, and 1:3. All samples were mixed gently and incubated for 5 minutes at room temperature prior to analyses. Dilutions of 1:22, 1:9, and 1:3 are approximately equivalent to in vivo dilution following volume resuscitation using 4, 10, and 30 mL/kg of fluids, respectively, taking into account a blood volume in adult dogs of approximately 90 mL/kg body weight.34 Statistical analysis All statistical analyses were performed with commercial software.p Data were analyzed for normality using D’Agostino–Pearson and Kolmogorov–Smirnov tests. 477

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Figure 3: Solutions by dilution ExTEM CFT: Comparison of in vitro effects on ROTEM ExTEM clot formation time (ExTEM CFT) of various solutions at different dilutions in canine blood. Results are displayed as median (bars) with 95% confidence intervals (error bars). The baseline measurement in whole blood is shown as a dashed line for reference. Significant differences between solutions at the same dilution are shown as a connector and asterisk.

Figure 4: Clinical Dilutions ExTEM CFT: Comparison of in vitro effects on ROTEM ExTEM clot formation time (ExTEM CFT) of various solutions in dilutions mimicking clinical doses in canine blood. Baseline measurements in whole blood are shown as a reference. Significant differences between solutions are shown as a connector and asterisk.

Far outliers were detected and excluded using Tukey and Grubbs double-sided (alpha 0.05) tests. Data for PFA analyses were reported as mean ± SD. Data for ROTEM analyses were reported as median (range) due

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to small sample size and normal as well as not normal distributed variables. A repeated measures ANOVA was performed to analyze differences between variables for PFA data. Where significant differences were

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Effects of volume expanders on hemostasis

Figure 5: Solutions by Dilution ExTEM MCF: Comparison of in vitro effects on ROTEM ExTEM maximal clot firmness (ExTEM MCF) of various solutions at different dilutions in canine blood. Results are displayed as median (bars) with 95% confidence intervals (error bars). The baseline measurement in whole blood is shown as a dashed line for reference. Significant differences between solutions at the same dilution are shown as a connector and asterisk.

Figure 6: Clinical Dilutions ExTEM MCF: Comparison of in vitro effects on ROTEM ExTEM maximum clot firmness (ExTEM MCF) of various solutions in dilutions mimicking clinical doses in canine blood. Baseline measurements in whole blood are shown as a reference. Significant differences between solutions are shown as a connector and asterisk.

found, pairwise comparisons were performed with Bonferroni correction. A Friedman Test was used to analyze differences for ROTEM data with post hoc multiple comparisons where significant differences were found.

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Dilutions mimicking clinical administration of 30 mL/kg 0.9% NaCl (0.9% NaCl at a ratio of 1:3), 4 mL/kg hypertonic saline (7.2% HTS and HH at a ratio of 1:22), and 10 mL/kg colloid (HES solutions at a ratio of 1:9)

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Figure 7: Solutions by Dilution FibTEM CT (1): Comparison of in vitro effects on ROTEM FibTEM clotting time (FibTEM CT) of various solutions at dilutions 1:22 and 1:9 in canine blood. Results are displayed as median (bars) with 95% confidence intervals (error bars). The baseline measurement in whole blood is shown as a dashed line for reference. Significant differences between solutions at the same dilution are shown as a connector and asterisk.

were compared using a Friedman Test and post hoc analysis. The level of significance was set to P < 0.05 throughout.

Results Twenty dogs fulfilled the inclusion criteria and were used in the results. One dog was excluded from the study because of a low platelet count (

Comparison of the in vitro effects of saline, hypertonic hydroxyethyl starch, hypertonic saline, and two forms of hydroxyethyl starch on whole blood coagulation and platelet function in dogs.

To compare the in vitro effects of hypertonic solutions and colloids to saline on coagulation in dogs...
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