Abstract

Background. Plasma substitutes such as hydroxyethyl starch (HES) and various dextrans may compromise the haemostatic system, thereby causing potentially dangerous bleeding. Whilst several mechanisms have been advanced to explain the nature of the coagulopathy induced by this colloid, there has been comparably little interest in devising ways to optimize haemostasis after a relative colloid overdose.

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 ×109 litre−1) and factor VIII (0.6 IU ml−1) produced insignificant changes in clot initiation, propagation, and in the clot strength. In contrast, ex vivo addition of a fibrinogen concentrate (1 g litre−1) 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.

Correction of acute blood loss often requires the use of plasma substitutes to maintain blood pressure and to preserve microvascular circulation. Sterile solutions of artificial colloids such as hydroxyethyl starch (HES) and dextrans are in widespread use for plasma volume expansion. Clinical reports indicate that prolonged infusion of plasma substitutes may result in an increased risk of spontaneous and potentially dangerous bleeding.12 Various experimental studies have demonstrated that plasma substitute supplementation induces a coagulopathy characterized by a compromised clot development,3 whereas the initiation phase of coagulation is unaffected.45 Haemostatic deficits are reportedly related to the type and composition of the plasma substitute. Thus, HES solutions with a high mean molecular weight (MW), a high content of hydroxyethyl motifs, and a high C2/C6 ratio, seemingly suppress the coagulation profile more than solutions composed of the more rapidly degradable low MW colloids.68 Although results and conclusions appear somewhat inconsistent,9 various mechanisms may be involved in the coagulopathy brought about by plasma substitutes, such as a reduction in the von Willebrand factor (vWF),10 acquired platelet dysfunction,11 reduced factor (F) VIII levels,12 and interaction with fibrinogen.13 However, at this time no studies have been published on possible approaches to reverse this coagulopathy.

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.

Materials and methods

Plasma substitutes

Equal volumes of four test solutions: isotonic saline 0.9% (Nycomed A/S, Roskilde, Denmark), HES 200/0.5 (Haes-Steril® 6%, Fresenius Kabi, Bad Homburg, Germany), HES 130/0.4 (Voluven® 6%, Fresenius Kabi), and dextran 70 (Macrodex®, Pharmalink AB, Upplands Väsby, Sweden) were investigated.

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 (FVIII–Octonativ®, 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 (27–55) 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 (DyCoDerivAn™, 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.

Fig 1

(a) Traditional thrombelastographic pattern and parameters. CT=clotting time; CFT=clot formation time; MCF=maximum clot firmness. (b) Velocity profile of coagulation expressed as first derivative of the thrombelastographic tracing. MaxVel=maximum velocity of clot formation; tMaxVel=time until maximum velocity.

Fig 1

(a) Traditional thrombelastographic pattern and parameters. CT=clotting time; CFT=clot formation time; MCF=maximum clot firmness. (b) Velocity profile of coagulation expressed as first derivative of the thrombelastographic tracing. MaxVel=maximum velocity of clot formation; tMaxVel=time until maximum velocity.

Procedure for ex vivo haemodilution

Citrated WB was diluted with each of the four test solutions. A volume of 100 µl test solution and 50 µl barbital buffer (sodium barbital 28 mM further diluted 3:4 in sodium chloride 150 mM) was pipetted into the roTEG cups. The roTEG cups were prewarmed and maintained at 37°C during the measurement process. Ten minutes after blood sampling, aliquots of 150 µl citrated WB were added into the roTEG cups. The roTEG analysis was performed as we have recently published,14 where the blood mixture was activated with 20 µl recombinant human tissue factor (Innovin, Dade Behring Marburg GmbH, Germany) prediluted 1:1000 in barbital buffer. The content of the roTEG cup was mixed by gently stirring with the pipette tip and recording of each of the four roTEG channels was initiated simultaneously with the addition of 20 µl calcium chloride (200 mM).

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 litre−1 and 0.6 IU ml−1, respectively, while addition of 20 µl of fibrinogen solution (20 mg ml−1) produced a 1 g litre−1 elevation in the existing fibrinogen level in the roTEG cup (normal plasma fibrinogen levels in healthy persons: 1.8–3.9 g litre−1).

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.

Results

Characterization of WB clot formation after addition of isotonic saline, HES, and dextran 70 (n=20)

The roTEG results and derived parameters are given in Table 1. Figure 2 illustrates characteristic dynamic WB coagulation profiles in undiluted WB14 as well as WB diluted with isotonic saline 0.9%, HES, and dextran 70. When compared with haemodilution by isotonic saline 0.9%, dilution by both HES solutions and dextran 70 did not affect the initiation phase of coagulation but diminished the profile of the propagation phase of WB clot formation as shown by a significant reduction in MaxVel and a prolongation of tMaxVel (Table 1 and Fig. 2). Furthermore, haemodilution with plasma expanders significantly depressed clot strength as indicated by a significant decrease in MCF (Table 1). We found no difference between HES 200/0.5 and HES 130/0.4. In addition, dextran 70 also suppressed clot development and clot strength but not to a significantly greater extent than the two HES solutions (Table 1).

Fig 2

Characteristic dynamic WB coagulation velocity profiles of undiluted WB as well as after in vitro haemodilution (55%) with isotonic saline, HES 130/0.4, HES 200/0.5 and dextran 70.

Fig 2

Characteristic dynamic WB coagulation velocity profiles of undiluted WB as well as after in vitro haemodilution (55%) with isotonic saline, HES 130/0.4, HES 200/0.5 and dextran 70.

Table 1

RoTEG analyses and derived parameters before (−fibr) and after (+fibr) addition of fibrinogen. Data are presented as mean (SD), n=20 in each group. CT=clotting time; CFT=clot formation time; MCF=maximal clot firmness; MaxVel=maximum velocity; tMaxVel=time until maximum velocity. Undiluted WB values from reference 14.

*

Significantly different from blood diluted with isotonic saline (P<0.05).

Significantly different from the same group before addition of fibrinogen (P<0.05)

 Isotonic saline0.9%
 
 HES 200/0.5
 
 HES 130/0.4
 
 Dextran 70
 
 Undiluted WB
 
 

 
−fibr
 
+fibr
 
−fibr
 
+fibr
 
−fibr
 
+fibr
 
−fibr
 
+fibr
 
Male
 
Female
 
roTEG® analyses           
    CT 335 (65) 206 (71) 321 (61) 349 (71) 353 (64) 348 (19) 342 (69) 329 (62) 354 (58) 322 (55) 
    CFT 258 (65) 169 (53) 283 (62) 227 (59) 472 (132)* 273 (56) 579 (215)* 308 (44) 141 (26)* 119 (28)* 
    MCF 45 (5.4) 53 (1.7) 38.6 (5.6)* 46 (5.3) 29 (5.4)* 38.5 (3.4) 36.6 (5.9)* 38 (3.5) 59 (3.7)* 61 (4.9)* 
Derived parameters           
    MaxVel 7.84 (1.8) 14.1 (0.6) 6.2 (1.6)* 9.82 (1.1) 6.3 (1.7)* 7.8 (1.8) 5.09 (1.7)* 7.2 (1.0) 14.8 (2.2)* 16.8 (3.2)* 
    tMaxVel 525 (88) 407 (40) 494 (69) 452 (230) 532 (79) 453 (219) 550 (106) 546 (96) 552 (75) 507 (70) 
 Isotonic saline0.9%
 
 HES 200/0.5
 
 HES 130/0.4
 
 Dextran 70
 
 Undiluted WB
 
 

 
−fibr
 
+fibr
 
−fibr
 
+fibr
 
−fibr
 
+fibr
 
−fibr
 
+fibr
 
Male
 
Female
 
roTEG® analyses           
    CT 335 (65) 206 (71) 321 (61) 349 (71) 353 (64) 348 (19) 342 (69) 329 (62) 354 (58) 322 (55) 
    CFT 258 (65) 169 (53) 283 (62) 227 (59) 472 (132)* 273 (56) 579 (215)* 308 (44) 141 (26)* 119 (28)* 
    MCF 45 (5.4) 53 (1.7) 38.6 (5.6)* 46 (5.3) 29 (5.4)* 38.5 (3.4) 36.6 (5.9)* 38 (3.5) 59 (3.7)* 61 (4.9)* 
Derived parameters           
    MaxVel 7.84 (1.8) 14.1 (0.6) 6.2 (1.6)* 9.82 (1.1) 6.3 (1.7)* 7.8 (1.8) 5.09 (1.7)* 7.2 (1.0) 14.8 (2.2)* 16.8 (3.2)* 
    tMaxVel 525 (88) 407 (40) 494 (69) 452 (230) 532 (79) 453 (219) 550 (106) 546 (96) 552 (75) 507 (70) 

vWF:RCoF and TT (n=20)

Analysis of mean (sd) vWF:RCoF showed no significant (P<0.05) change with HES 200/0.5 (0.43 (0.12) IU ml−1), HES 130/0.4 (0.44 (0.14) IU ml−1), and dextran 70 (0.41 (0.15) IU ml−1) when compared with dilution with saline 0.9% (0.50 (0.20) IU ml−1). Similarly, analysis of the TT revealed no significant (P<0.05) changes with HES 200/0.5 (20 (1.7) s), HES 130/0.4 (19.6 (1.8) s), and dextran 70 (19.6 (1.8) s) when compared with dilution with saline 0.9% (20.3 (1.7) s).

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 litre−1. 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.

Fig 3

(ad) Characteristic velocity profiles of whole blood clot formation after in vitro haemodilution (55%) with HES 130/0.4, 200/0.5 and dextran 70. Before (grey) and after (black) ex vivo addition of the fibrinogen concentrate.

Fig 3

(ad) Characteristic velocity profiles of whole blood clot formation after in vitro haemodilution (55%) with HES 130/0.4, 200/0.5 and dextran 70. Before (grey) and after (black) ex vivo addition of the fibrinogen concentrate.

Composition of the fibrinogen concentrate

Immune electrophoresis revealed that the majority of protein present in the fibrinogen concentrate actually was fibrinogen. However, there were traces of other proteins (e.g. albumin and coagulation FXIII). PT measurements with 1:1 mixtures of the fibrinogen concentrates and FII, FVII, and FX depleted plasma revealed no changes, showing that no major contamination with these coagulation factors was present. The fibrinogen concentrate contained 1.72 IU ml−1 of FXIII, thus the final amount of FXIII added was 0.01 IU ml−1 in the ex vivo haemostatic intervention studies.

Discussion

Several clinical and experimental investigations revealed that haemodilution with plasma substitutes may result in an iatrogenic haemorrhagic diathesis.1 The haemostatic dysfunction has been ascribed to a wide range of possible mechanisms such as compromised activity of vWF, FVIII, and platelets. The main purposes of this study has been to characterize the shift in WB coagulation profiles after extensive haemodilution induced by various plasma substitutes and to explore the haemostatic effect of a variety of available candidate haemostatic agents. In the present study, the entire coagulation process was evaluated using a thrombelastographic method employing activation with tissue factor representing the physiological initiator of coagulation.15 Analysis of recordings from the instrument included novel data processing of the thrombelastographic signal,14 which improved the interpretation of changes in coagulation dynamics. The time-course of the velocity profile of WB clot formation appears highly similar to the thrombin generation profiles of plasma reported by Hemker colleagues.16 Consequently, the velocity profiles may constitute an indirect assessment of changes in thrombin generation. Other investigations have demonstrated that modest haemodilution by around 20–30% with crystalloids induces a procoagulant course of coagulation.1719 However, in contrast to our previous investigations of undiluted WB,14 the present study demonstrated that profiles of continuous WB coagulation were significantly compromised by in vitro haemodilution to ∼55% using HES 200/0.5, HES 130/0.4, and dextran 70. The plasma substitutes tested significantly affected the coagulation profile more profoundly than haemodilution with isotonic saline. In this respect, no significant variation was found amongst the HES solutions and dextran 70. However, dextran 70 tended to produce the most pronounced inhibition of coagulation. The roTEG results and the derived parameters revealed that the initiation phase of WB clot formation was unchanged while the MaxVel in the propagation phase of clot formation was decreased. Furthermore, a significantly diminished clot strength was detected when colloids were present as demonstrable by a reduced MCF. The amplitude of the MCF has been reported to correlate positively with platelet count and the concentration of fibrinogen.20 Thus, the induced coagulopathy was thought to be attributable to dysfunction of platelets or fibrinogen. Based on previously reported studies and hypotheses generated in our laboratory, we explored various candidate haemostatic agents. Ex vivo addition of washed platelets and FVIII, respectively, produced a slightly earlier initiation of clot formation but failed to improve the reduced MaxVel of clot development in addition to clot strength, however, with the proviso that this observation is based on few samples. In contrast, addition of a commercially available fibrinogen concentrate reversed the coagulopathy to a level approaching the effect of dilution with isotonic saline. As measurements of the PT did not alter after mixture of the fibrinogen concentrate with FII, FVII, and FX depleted plasma, respectively, it was assumed that the presence of extra fibrinogen molecules explain the haemostatic enhancement. In addition, immune electrophoresis demonstrated that fibrinogen was the dominant protein present in the concentrate. Furthermore, as only small amounts of FXIII were present, fibrinogen was assessed as the protein component responsible for the observed haemostatic effect of the fibrinogen concentrate.

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 dose–response relationships.

Special thanks to Niels Trolle Andersen, Department of Biostatistics, Aarhus University for statistical assistance.

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Author notes

1Department of Anaesthesiology, Aarhus University Hospital, Aarhus Sygehus, Denmark. 2Center for Haemophilia and Thrombosis, Department of Clinical Biochemistry, Aarhus University Hospital, Skejby Sygehus, Denmark

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