1 Department of Anaesthesiology, Aarhus University Hospital, Aarhus Sygehus, Denmark. 2 Center for Haemophilia and Thrombosis, Department of Clinical Biochemistry, Aarhus University Hospital, Skejby Sygehus, Denmark
* Corresponding author. E-mail: ingerslev{at}ki.au.dk
Accepted for publication October 12, 2004.
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Abstract |
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Methods. Real-time whole blood (WB) clot formation profiles were recorded using a thrombelastographic method employing activation with tissue factor. The coagulation tracings were transformed into dynamic velocity profiles of WB clot formation. WB from healthy individuals (n=20) was exposed to haemodilution of 55% with isotonic saline, HES 200/0.5, HES 130/0.4, and dextran 70, respectively. Possible modalities for improvement of the induced coagulopathy were explored, in particular ex vivo addition of a fibrinogen concentrate.
Results. WB coagulation profiles changed significantly with decreased clot strength, and a compromised propagation phase of clot formation. The duration of the initiation phase of WB coagulation was unchanged. No statistical differences were detected amongst the HES solutions and dextran 70. However, dextran 70 returned a more suppressed clot development and strength compared with the HES solutions. Ex vivo haemostatic addition of washed platelets (75 x109 litre1) and factor VIII (0.6 IU ml1) produced insignificant changes in clot initiation, propagation, and in the clot strength. In contrast, ex vivo addition of a fibrinogen concentrate (1 g litre1) improved the coagulopathy induced by all of the three individual plasma expanders tested.
Conclusion. Coagulopathy induced by haemodilution with either HES 200/0.5, HES 130/0.4, and dextran 70 may be improved by fibrinogen supplementation.
Keywords: blood, coagulation ; blood, haemodilution ; blood, replacement ; measurement techniques, thrombelastograph
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Introduction |
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In the present study we report on the effect of ex vivo haemodilution with isotonic saline (0.9%), HES 200/0.5 (Haes Steril®), HES 130/0.4 (Voluven®), and dextran 70 (Macrodex®) as assessed using a thrombelastographic model investigating continuous whole blood (WB) clot formation initiated by tissue factor (TF), and a new method for data processing of the thrombelastographic signal.14 Furthermore, mechanisms involved in the coagulopathy after haemodilution of platelet poor plasma by the volume expanders were studied by recording vWF: ristocetin cofactor ratio (vWF:RCoF) activity, thrombin time, and the activity of coagulation FXIII. To address possible mechanisms underlying the coagulopathy indicated by the plasma substitutes, we studied the haemostatic effect of ex vivo addition of normal platelets, highly purified FVIII and ex vivo supplementation with a commercially available fibrinogen concentrate.
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Materials and methods |
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Haemostatic components
The following haemostatic components were examined in this study: washed platelets purified using a Ficoll® gel (Amersham Biosciences, Uppsala, Sweden) technique following manufacturer's recommendations, a monoclonal antibody purified FVIII (FVIIIOctonativ®, Biovitrum, Stockholm, Sweden), and a pasteurized human fibrinogen concentrate (Haemocompletan®, Aventis Behring GmbH, Marburg, Germany).
Patients
In total, 20 healthy patients, 11 females and 9 males, with a mean (range) age of 35 (2755) yr, a mean (SD) body weight of 66 (10) kg, and a height of 171 (9.5) cm volunteered for venipuncture. None of the participants had received medication with acetyl-salicylic acid or any non-steroid anti-inflammatory drugs 7 days before blood sampling. Six of the 20 volunteers provided an additional blood sample for further investigations on the haemostatic response to fibrinogen concentrate. Another two volunteers provided blood samples used in experiments exploring the haemostatic effect of addition of platelets and FVIII.
Blood sampling
A smooth venipuncture was performed employing minimum stasis and a 21-gauge butterfly needle. Blood samples were drawn into citrated Venoject® tubes (Terumo Europe, Leuven, Belgium (0.129 M: 3.8 w/v %)) mixing one part of citrate with nine parts of blood, discarding the first tube aspirated.
Thrombelastographic coagulation analysis
All analyses were performed using a roTEG® Thrombelastograph Coagulation Analyser (Pentapharm, Munich, Germany). According to manufacturer's procedure, thrombelastographic parameters; clotting time (CT), clot formation time (CFT) and maximal clot firmness (MCF) were recorded (Fig. 1A). The raw roTEG analyser signal was further analysed using a novel software program (DyCoDerivAnTM, AvordusoL, Risskov, Denmark) for calculation of dynamic coagulation parameters such as MaxVel and t,MaxVel (Fig. 1B). MaxVel is the peak rate of clot formation and tMaxVel corresponds to the time until occurrence of maximum velocity. Thus, the CT defines the initiation phase of coagulation and the MaxVel and tMaxVel concur with the propagation phase of clot formation, while the MCF expresses a measure of the final clot strength. The MCF is equivalent to the area under the velocity curve.
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Final reaction volume in the roTEG cup of 340 µl produced a relative degree of haemodilution of 55%. All analyses were processed in duplicate for at least 90 min.
Procedure for ex vivo addition of haemostatic components
Samples used to examine the effect of addition of the haemostatic components were haemodiluted as described above and platelets (20 µl), FVIII concentrate (25 µl) or fibrinogen (20 µl) was added. The final concentration in the roTEG cup after addition of platelets and FVIII corresponded to 45 mia litre1 and 0.6 IU ml1, respectively, while addition of 20 µl of fibrinogen solution (20 mg ml1) produced a 1 g litre1 elevation in the existing fibrinogen level in the roTEG cup (normal plasma fibrinogen levels in healthy persons: 1.83.9 g litre1).
Analysis of vWF:ristocetin cofactor ratio (vWF:RCoF)
vWf:RCoF analysis was performed in plasma from each of the 20 volunteers after haemodilution with each of the four test solutions. All measurements were performed on a Behring Coagulation Timer (BCT) Coagulation Analyzer (Dade Behring). A total sample volume of 40 µl was incubated for 15 s with 1 µl of imidazol buffer (50 mM, pH=7.3) and measurements were started following addition of 150 µl Behring Coagulation (BC) von Willebrand Reagent (Dade Behring) containing stabilized platelets and ristocetin.
Analysis of the thrombin time
The thrombin time (TT) was analysed on a BCT Coagulation Analyzer (Dade Behring) using bovine thrombin (BC Thrombin Reagent, Dade Behring) as the test reagent.
Analyses performed with the fibrinogen concentrate
The commercially available fibrinogen concentrate was tested for purity by 2-D immune electrophoresis. In the first dimension, the fibrinogen concentrate was run on a 1% agarose gel in a tris-barbital buffer (98 mM, pH=8.6). The second dimension gel containing rabbit anti-total human plasma proteins was aligned side-to-side with the first dimension gel on a glass plate, and electrophoresis current was directed from first to second gel. Coomassie blue was used to stain electrophoretic bands.
Prothrombin time (PT) was performed by mixing the fibrinogen concentrate 1:1 with plasma samples depleted in FII, FVII, and FX. The PT test reagent used was a commercial combined reagent (Nycomed A/S, Oslo, Norway) containing rabbit brain tissue factor and excess bovine fibrinogen and FV. The level of FXIII in the fibrinogen concentrate was measured photometrically using a chromogenic substrate (Berichrom® FXIII, Dade Behring). Before determination of FXIII, the fibrinogen concentrate required 1:5 dilution to overcome interference with the high level of fibrinogen. Results were corrected for this dilution.
Data analysis and statistics
All statistical analyses were performed using the statistical program SPSS® version 10.0 (SPSS Inc., Chicago, IL, USA). The effect of haemodilution with plasma expander in comparison with haemodilution using isotonic saline 0.9% was assessed using a paired Student's t-test. The between-group differences were compared using one-way ANOVA. P<0.05 was considered statistically significant.
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Results |
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Effect of ex vivo haemostatic intervention with platelets and FVIII (n=2)
When washed platelets were added, a shortened initiation phase of clot formation was seen but clot development and clot strength were not improved. Ex vivo addition of FVIII did not change clot development or clot strength, but resulted in a slightly earlier initiation of clot formation (data not shown).
Effect of ex vivo haemostatic intervention with fibrinogen concentrate (n=6)
Figure 3AD shows characteristic profiles after ex vivo spiking with the fibrinogen concentrate corresponding an addition of extra 1 g litre1. In the presence of the fibrinogen concentrate clot strength and propagation phase of clot formation was increased as shown by significant changes in MCF and MaxVel (Table 1). Thus, when compared with reference values of MaxVel and MCF after haemodilution with isotonic saline 0.9%, ex vivo addition of fibrinogen completely, or partially, improved the coagulopathy induced by all of the plasma expanders investigated. In contrast, in blood prediluted with colloids, the fibrinogen concentrate did not alter the initiation phase of coagulation.
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Discussion |
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In conclusion, this study suggests that coagulopathy induced by haemodilution with HES 200/0.5, HES 130/0.4, and dextran 70 may be improved by a fibrinogen concentrate. It further appears that addition of platelets and FVIII did not improve the depressed clotting profiles. Clinical in vivo studies are required to show whether our haemostasis model may be applied to the clinical setting and assess possible doseresponse relationships.
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Acknowledgments |
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References |
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2 Lockwood DN, Bullen C, Machin SJ. A severe coagulopathy following volume replacement with hydroxyethyl starch in a Jehovah's Witness. Anaesthesia 1988; 43: 3913[ISI][Medline]
3 Mortier E, Ongenae M, De Baerdemaeker L, et al. In vitro evaluation of the effect of profound haemodilution with hydroxyethyl starch 6%, modified fluid gelatin 4% and dextran 40 10% on coagulation profile measured by thromboelastography. Anaesthesia 1997; 52: 10614[ISI][Medline]
4 Egli GA, Zollinger A, Seifert B, Popovic D, Pasch T, Spahn DR. Effect of progressive haemodilution with hydroxyethyl starch, gelatin and albumin on blood coagulation. Br J Anaesth 1997; 78: 6849
5 Petroianu GA, Liu J, Maleck WH, Mattinger C, Bergler WF. The effect of in vitro hemodilution with gelatin, dextran, hydroxyethyl starch, or Ringer's solution on Thrombelastograph. Anesth Analg 2000; 90: 795800
6 Entholzner EK, Mielke LL, Calatzis AN, Feyh J, Hipp R, Hargasser SR. Coagulation effects of a recently developed hydroxyethyl starch (HES 130/0.4) compared to hydroxyethyl starches with higher molecular weight. Acta Anaesthesiol 2000; 44: 111621[CrossRef][ISI]
7 Jamnicki M, Zollinger A, Seifert B, Popovic D, Pasch T, Spahn DR. Compromised blood coagulation: an in vitro comparison of hydroxyethyl starch 130/0.4 and hydroxyethyl starch 200/0.5 using thrombelastography. Anesth Analg 1998; 87: 98993[Abstract]
8 Langeron O, Doelberg M, Ang ET, Bonnet F, Capdevila X, Coriat P. Voluven, a lower substituted novel hydroxyethyl starch (HES 130/0.4), causes fewer effects on coagulation in major orthopedic surgery than HES 200/0.5. Anesth Analg 2001; 92: 85562
9 Stoll M, Treib J, Schenk JF, et al. No coagulation disorders under high-dose volume therapy with low-molecular-weight hydroxyethyl starch. Haemostasis 1997; 27: 2518[ISI][Medline]
10 Jonville-Bera AP, Autret-Leca E, Gruel Y. Acquired type I von Willebrand's disease associated with highly substituted hydroxyethyl starch. N Engl J Med 2001; 345: 6223
11 Stogermuller B, Stark J, Willschke H, Felfernig M, Hoerauf K, Kozek-Langenecker SA. The effect of hydroxyethyl starch 200 kD on platelet function. Anesth Analg 2000; 91: 8237
12 de Jonge E, Levi M, Buller HR, Berends F, Kesecioglu J. Decreased circulating levels of von Willebrand factor after intravenous administration of a rapidly degradable hydroxyethyl starch (HES 200/0.5/6) in healthy human subjects. Intensive Care Med 2001; 27: 18259[CrossRef][ISI][Medline]
13 Strauss RG, Stump DC, Henriksen RA, Saunders R. Effects of hydroxyethyl starch on fibrinogen, fibrin clot formation, and fibrinolysis. Transfusion 1985; 25: 2304[CrossRef][ISI][Medline]
14 Sorensen B, Johansen P, Christiansen K, Wöelke M, Ingerslev J. Whole blood coagulation thrombelastographic profiles employing minimal tissue factor activation. J Thromb Haemost 2003; 1: 5518[CrossRef][ISI][Medline]
15 Rapaport SI, Rao LV. The tissue factor pathway: how it has become a prima ballerina. Thromb Haemost 1995; 74: 717[ISI][Medline]
16 Hemker HC, Giesen PL, Ramjee M, Wagenvoord R, Beguin S. The thrombogram: monitoring thrombin generation in platelet-rich plasma. Thromb Haemost 2000; 83: 58991[ISI][Medline]
17 Tobias MD, Wambold D, Pilla MA, Greer F. Differential effects of serial hemodilution with hydroxyethyl starch, albumin, and 0.9% saline on whole blood coagulation. J Clin Anesth 1998; 10: 36671[CrossRef][ISI][Medline]
18 Ruttmann TG, James MF, Viljoen JF. Haemodilution induces a hypercoagulable state. Br J Anaesth 1996; 76: 41214
19 Ruttmann TG, James MF, Lombard EH. Haemodilution-induced enhancement of coagulation is attenuated in vitro by restoring antithrombin III to pre-dilution concentrations. Anaesth Intensive Care 2001; 29: 48993[ISI][Medline]
20 Oshita K, Az-ma T, Osawa Y, Yuge O. Quantitative measurement of thromboelastography as a function of platelet count. Anesth Analg 1999; 89: 2969