Influence of different colloids on molecular markers of haemostasis and platelet function in patients undergoing major abdominal surgery

I. Hüttner1, J. Boldt1, G. Haisch1, St. Suttner1, B. Kumle1 and H. Schulz2

1Department of Anaesthesiology and Intensive Care Medicine, and 2Clinic of Surgery, Klinikum der Stadt Ludwigshafen, Bremserstr. 79, D-67063 Ludwigshafen, Germany*Corresponding author

Accepted for publication: 16 April 2000


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Synthetic colloids have been reported to cause haemorrhagic complications. The effects of perioperative volume replacement with 4% gelatin (n=20), 6% low-molecular weight (LMW) hydroxyethyl starch (HES) (Mw: 70 000 dalton; HES 70/0.5; n=20) and 6% medium-molecular weight (MMW) HES (Mw: 200 000 dalton; HES 200/0.5; n=20) on haemostasis were assessed in patients undergoing major abdominal surgery. Volume was administered to keep central venous pressure (CVP) between 10 and 14 mm Hg. Conventional global coagulation tests, molecular markers of coagulation, and platelet function (using a platelet function analyser (PFA-100TM) with ADP as inductor) were monitored prior to surgery (T0), at the end of surgery (T1), 4 h after the end of surgery (T2), and on the morning of the first postoperative day (T3). Significantly more gelatin (2900 (SD 320) ml) than HES 200 (2150 (312) ml) was given during the study period. Bleeding and the use of allogeneic blood–blood products were similar in all groups. Markers of thrombin generation (F1+2), of thrombin neutralization (TAT III complex), and of fibrin formation and its degradation (D-dimer) increased significantly during and after surgery without showing significant group differences. Factor VIII and von Willebrand factor (vWF) also increased in all groups beyond the normal range, showing the significantly highest increase in the gelatin-treated group (VIII: from 173 (36) to 266 (33) U dl–1; vWF: from 164(33) to 238 (31) U dl–1). Platelet function remained within the normal range and without group differences throughout the study period. We can conclude that all three solutions can be used safely in patients undergoing major abdominal surgery with regard to the haemostatic process.

Br J Anaesth 2000; 85: 417–23

Keywords: surgery; haemorrhage; blood, platelets; measurement techniques


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
During surgery, absolute or relative blood volume deficits often occur either due to bleeding (absolute hypovolaemia) or vasodilation mediated by various vasodilating substances (relative hypovolaemia). The most important objective of volume therapy in this situation is to maintain a stable haemodynamic state and tissue perfusion without inducing side effects. The inherent risk of transmission of viral and immunological diseases has forced us to reduce the use of allogeneic blood. A variety of synthetic colloidal solutions have been introduced to enhance blood volume. The ideal regimen of volume replacement has been the focus of debate for several years. In spite of the widespread use of synthetic colloids in several countries, there is still considerable concern that synthetic colloids may have adverse effects on coagulation.13 Some studies suggest that colloids [e.g. gelatin or hydroxyethyl starch (HES)] are effective and safe substitutes for blood loss without relevant adverse effects on coagulation.4 5 Others showed increased bleeding tendency with colloids and suggested that they should be avoided especially in patients at risk of increased bleeding.6 Most of these studies, however, have assessed only conventional coagulation data. Serial monitoring of molecular markers of haemostasis is more helpful for the diagnosis of coagulation disorders.7 The present study was designed to evaluate the influence of intraoperative volume replacement with three different colloids in patients undergoing major abdominal surgery on the following markers of haemostasis: prothrombin fragment F1 + 2 (as a marker of thrombin generation); TAT III (as a marker of thrombin neutralization); D-dimer (as a marker of fibrin formation and its degradation); and the factor VIII–von Willebrand factor (vWF) system (as a marker of endothelial injury). Additionally, platelet function was assessed using the PFA-100TM system in which coagulation is simulated close to normal conditions.8 9


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The study was approved by the Ethics Committee of the hospital and all patients gave written informed consent. Sixty consecutive patients undergoing major abdominal surgery were included in the study. Exclusion criteria were cardiac insufficiency (NYHA class III–IV), renal insufficiency (serum creatinine >2 mg dl–1), liver dysfunction (aspartate aminotransferase >40 U l–1, alanine aminotransferase >40 U l–1), preoperative anaemia (haemoglobin (hgb) <10.0 g dl–1), preoperative coagulation abnormalities (platelet count <100 nl–1; activated partial thromboplastin time (aPTT) >70 s; fibrinogen <2 g dl–1; anti-thrombin III <40%), non-steroidal therapy or use of cyclooxygenase inhibitors. Standard anti-thrombotic prophylaxis using low-molecular weight heparin administered s.c. the evening before surgery was used in all patients.

According to preoperative randomization (using blinded envelopes), the patients were prospectively separated into three groups (each group n=20). Patients received either 4% modified gelatin solution (Gelafundin (B. Braun, Melsungen, Germany)), a low-molecular weight (LMW) HES solution (mean molecular weight (Mw) 70 000 dalton, degree of substitution (DS) 0.5; 6% HES 70/0.5 (Rheohes, B. Braun)), or a medium-molecular weight (MMW) HES (Mw 200 000 dalton, DS 0.5; 6% HES 200/0.5 (Hemohes, B. Braun)). Volume was given to maintain central venous pressure (CVP) between 10 and 14 mm Hg. Packed red blood cells (PRBC) were administered when the haemoglobin was <9 g dl–1. Fresh frozen plasma (FFP) was given only to maintain haemostasis (when aPTT >70 s, fibrinogen <2 g dl–1, antithrombin III <40%, and bleeding occurs). Platelets were infused when the platelet count was <30x109 litre–1. Ringer’s lactate was given to compensate fluid loss by sweating, gastric tubes and urine output, or as a solvent for drugs (e.g. antibiotics). Ringer’s lactate 500 ml h–1 was administered routinely in all patients during surgery.

The patients were premedicated with lorazepam 2.0 mg. Anaesthesia was induced in all groups with fentanyl 3 µg kg–1, thiopental 5 mg kg–1 and atracurium 0.5 mg kg–1. Anaesthesia was maintained by titrating sufentanil, atracurium and isoflurane as indicated clinically. The lungs of all patients were mechanically ventilated with 60% N2O in oxygen to keep the SaO2 >95% (continuous oximetry) and the end-expiratory CO2 between 35 and 40 mm Hg (continuous capnography). To maintain normothermia during surgery, a rewarming system (WarmtouchTM) and fluid warmers were used. After surgery, the patients were transferred either to an intensive care unit (ICU) or to an intermediate care unit. Perioperative monitoring included continuous arterial pressure measurement (radial artery), ECG, pulse oximetry, CVP, body temperature (oesophageal), urine output and arterial blood–gas analysis. The anaesthetists who were responsible for the patients’ management were not involved in the study and blinded to the grouping.

Coagulation measurements
From central venous blood samples, standard coagulation data (antithrombin III (AT III), fibrinogen, platelet count, activated partial thromboplastin time (aPTT)) were measured using routine laboratory tests. D-dimer (turbidimetric method, Roche Diagnostics, Mannheim, Germany, inter-assay coefficient of variability in normal ranges (CV): <10%), F1+2 (ELISA, Dade/Behring, Marburg, Mannheim, CV: 6–13%), thrombin/anti-thrombin III (TAT; ELISA, Dade/Behring, Marburg, CV: 6–9%), factor VIII activity (one-stage clotting assay; Roche Diagnostics, Mannheim, Germany, CV: 7%), von Willebrand factor (vWF; turbidimetric method, Roche Diagnostics, Mannheim, Germany, CV: <10%), collagen-binding activity of von Willebrand factor (vWF: CBA; sandwich ELISA, Immuno-Diagnostika, Heidelberg, Germany, CV: 17%) were also measured from the blood samples. Platelet function was measured using a Platelet Function Analyser PFA-100TM system (Dade/Behring, Marburg, Germany). The time required for the platelet plug to occlude the aperture is called the ‘closure time’ and is indicative of platelet function.8 When the PFA-100TM data using ADP as an inductor are abnormal, it is likely that patients have a platelet defect that impairs primary haemostasis.9 The normal range for PFA-100TM system using ADP as an inductor has been determined to be 77–130 s.9 Results from all coagulation data represent the mean from duplicate measurements. Blood sampling was performed at T0 (prior to surgery), T1 (end of surgery), T2 (4 h after surgery) and T3 (first postoperative day).

Statistics
SPSS/PC+ software (V4.0 SPSS, Chicago, USA) was used for statistical analysis. All measured and calculated data were normally distributed (tested by the Kolmogorov–Smirnov test) and all data are presented as mean (SD). Perioperative data were analysed using the Student’s t-test, Fisher’s exact test, or Wilcoxon rank sum test as appropriate. A two-way analysis of variance for repeated measurements (ANOVA, followed by Scheffe’s test) was used to determine the effects of group, time and group–time interactions for each measured coagulation variable. In the presence of significant time differences, pair-wise comparisons against baseline values were evaluated using multiple paired t-tests. P-values <0.05 were considered significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patient characteristics and data from the perioperative period are presented in Table 1. The use of PRBC and FFP was similar in all groups (Table 1). Haemodynamic variables and oesophageal temperature did not differ between the groups (Table 2). Similar volumes of crystalloid solutions were infused perioperatively (Table 2), but significantly more gelatin than HES 200/0.5 was needed to keep the CVP between 10 and 14 mm Hg (Table 2). Blood loss was comparable at each data point for all groups (Table 2). The degree of haemodilution (haemoglobin) was comparable among the groups throughout the study period (Table 3). Standard coagulation data (platelet count, aPPT, fibrinogen, AT III) did not show significant group differences within the study period (Table 3).


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Table 1 Patient‘s characteristics and perioperative data. Mean (SD); ICU, intensive care unit; PRBC, packed red blood cells; FFP, fresh frozen plasma
 

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Table 2 Infused volume (cumulative), haemodynamics and temperature. Mean (SD); MAP, mean arterial blood pressure; CVP, central venous pressure; POD, postoperative day. #P<0.05 different to the HES 200/0.5 group
 

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Table 3 Standard laboratory data. Mean (SD); POD, postoperative day. *P<0.05 different to baseline
 
F1+2 increased significantly from baseline values in all patients (Fig. 1). At the end of the study, F1+2 plasma levels had almost returned to baseline. Starting from normal concentrations at baseline (<1 mg l–1), D-dimer levels increased significantly without showing group differences (Fig. 1). TAT complex was also almost normal at baseline (<4 µg l–1), increased significantly beyond normal levels in all three groups, and was still elevated beyond normal at the end of the study in all patients (Fig. 1). Factor VIII activity and vWF were similar at baseline in all groups (Fig. 2). In the gelatin-treated patients, factor VIII (from 173 (36) to 266 (33) U dl–1) and vWF (from 164 (33) to 238 (31) U dl–1) increased significantly more than in the HES 70- and HES 200-treated groups (Fig. 2). On the morning of the first postoperative day, both factor VIII and vWF were significantly higher than at baseline in all groups, but there were no differences between groups. vWF : CBA increased similarly (P=0.07) in all three groups (Fig. 2). The platelet function test using ADP as an inductor remained within the normal range and without differences between the groups throughout the study period (Fig. 3).



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Fig 1 Changes of D-dimer (normal values: <0.5 mg l–1), F1+2 (normal values: <1.0 nmol litre–1), and thrombin/anti-thrombin III complex (TAT; normal values: <4.0 µg litre–1). Mean (SD); *P<0.05 different from baseline values. T0 (prior to surgery), T1 (end of surgery), T2 (4 h after surgery) and T3 (first postoperative day).

 


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Fig 2 Changes of factor VIII (normal values: 60–150 U dl–1), von Willebrand factor (vWF; normal values: 60–160 U dl–1), and collagen-binding activity of von Willebrand factor (vWF-CBA; normal values: 0.6–1.8 U ml–1). Mean (SD); *P<0.05 different from baseline values; +P<0.05 different to the other groups. T0 (prior to surgery), T1 (end of surgery), T2 (4 h after surgery) and T3 (first postoperative day).

 


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Fig 3 Changes of ‘closing time’ of the Platelet Function Analyser PFA-100TM using ADP as an inductor (normal values: 77–130 s). Mean (SD); *P<0.05 different to the other group. T0 (prior to surgery), T1 (end of surgery), T2 (4 h after surgery) and T3 (first postoperative day).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The major result of the present study was that, despite infusing more than 2500 ml of each colloid, blood loss, use of allogeneic blood–blood products and coagulation markers did not differ between the groups. While no differences in blood loss were found in the present study, a higher blood loss in HES- (6% HES 200/0.5) than in gelatin-treated patients undergoing primary total hip replacement has been reported.11 Other reports have used first generation, high-molecular weight (HMW), highly substituted HES (6% HES 450/0.7 (e.g. hetastarch)).1 6 In healthy volunteers, a decrease in factor VIII and a prolongation of aPTT were noticed after 1000 ml of HMW-HES.12 Patients in whom large amounts of HMW-HES (>1000 ml) were used had a decrease in factor VIII : C and vWF : AG.13 After infusion of 1500 ml of HMW-HES in surgical patients, Conroy et al.14 showed a significant decrease of factor VIII : C levels (69% of baseline). Although HES with lower MW and lower DS seems to cause fewer effects on the factor VIII system,2 15 16 negative effects of LMW- and MMW-HES on haemostasis have also been shown. In healthy volunteers, Kapiotis et al.17 demonstrated a specific lowering effect on factor VIII : C concentrations after 500 ml of MMW-HES (200 000 dalton; DS 0.5). They also found no specific influence on systemic activation of blood coagulation (measured by TAT plasma levels) and the fibrinolytic system (e.g. D-dimer).

Some in vitro studies have described advantages of gelatin compared to HES with regard to coagulation.1820 However, in healthy volunteers, 1000 ml of gelatin (Gelofusin) resulted in a significant decrease in vWF (–32%) most likely due to a binding of vWF to gelatin at its binding sites.21 Additionally, TAT (–45%) and F1+2 (–40%) decreased significantly more than could be expected by haemodilution, indicating that gelatin in healthy volunteers resulted in a significant impairment of primary haemostasis and thrombin generation.21 The defect in primary haemostasis appears to be related to a gelatin-induced reduction in von Willebrand factor. In an in vitro study, Tabuchi et al.22 found that gelatin reduced the velocity and the extent of ristocetin-induced platelet aggregation, whereas aggregation by ADP was not affected. They concluded that gelatin interfered with plasma vWF, not with platelet-vWF or with platelet GpIb.

The results of clinical trials may differ from in vitro studies. In thromboelastograph (TEG) studies, Karoutsos et al.23 revealed no hypocoagulability after moderate doses of gelatin, HES (200/0.5) or albumin (mean doses 1300–1800 ml). They found a hypercoagulability state only after administration of gelatin. Studies in volunteers and in vitro studies reproduce poorly the multiple in vivo interactions leading to coagulation.23 After surgery, a hypercoagulable state may develop,10 but in all in vitro studies this natural hypercoagulability is absent. In the present study, TAT, F1+2, D-dimer and factor VIII increased significantly during the study period, indicating thrombin generation and reactive hyperfibrinolyis during and after surgery. Hobisch-Hagen and colleagues24 showed that global tests of coagulation (PT, Quick, aPTT) were only slightly influenced by gelatin used in orthopaedic patients undergoing acute normovolaemic haemodilution. They found, however, increased concentrations of activation markers for coagulation (F1+2), TAT and fibrinolysis (D-dimer). The influence of intravascular volume replacement with synthetic colloids on platelet function is another subject of debate. Infusion of HMW-HES in patients undergoing orthopaedic surgery resulted in a clear ‘trend’ towards decreased platelet aggregation.5 In a case report, a reduced level of all three main factor VIII parameters (VIII : C, vWF, vWFag) was found in a patient after infusion of HMW-HES.25 With reduced VIIIR-RCo there is reduced binding to platelet membrane receptor proteins GPIb and GPIIb/IIIa which results in a decreased platelet adhesion. After large doses of HMW-HES, the platelets appeared swollen and platelet adhesion was reduced.26 Thurner27 observed a significant decrease of ristocetin-induced platelet aggregation in gelatin-treated patients, whereas in a HES 200/0.5-treated group, significantly less change in platelet function was found. Evans and co-workers28 showed that gelatin was associated with varying effects on platelet function: Haemaccel® resulted in more severe disturbances of platelet aggregation than Gelofusine® most likely due to its higher Ca2+ concentration. In the present study, we used the PFA-100TM system to assess platelet function. This system uses whole blood and thus provides a more realistic environment to the aggregation of platelets than other tests (e.g. using platelet aggregometry with platelet-rich plasma).8 The ‘closure time’ increased slightly during the study, indicating reduced platelet function. This, however, was within the normal range and without differences between the groups. It is summarized that in patients undergoing major abdominal surgery infusing moderate amounts of gelatin, HES 70/0.5 or HES 200/0.5 did not differ with regard to bleeding tendency and use of allogeneic blood. Serially measured molecular markers of haemostasis and indicators of platelet function revealed no relevant differences among the three groups. All three regimens of volume replacement appear to be safe concerning haemostasis in these patients.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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