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Keywords:

  • blood coagulation factors;
  • coagulopathy;
  • fibrinogen;
  • hemodilution;
  • hydroxyethyl starch

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Summary. Background: The biochemical mechanisms causing dilutional coagulopathy following infusion of hydroxyethyl starch 130/0.4 (HES) are not known in detail. Objectives: To give a detailed biochemical description of the mechanism of coagulopathy following 30%in vivo dilution with HES, to present a systematic ex vivo test of various hemostatic agents, and to investigate the hypothesis that acquired fibrinogen deficiency constitutes the most important determinant of the coagulopathy. Methods: Dynamic whole blood clot formation assessed by thromboelastometry, platelet count, thrombin generation, and the activities of von Willebrand factor, coagulation factor II, FVII, FVIII, FIX, FX and FXIII were measured in 20 bleeding patients enrolled in a prospective clinical study investigating in vivo substitution of blood loss with HES up to a target level of 30%. Thromboelastometry parameters were further evaluated after ex vivo spiking experiments with fibrinogen, prothrombin complex concentrate (PCC), FXIII, activated recombinant FVIIa (rFVIIa), fresh frozen plasma, and platelets. Results: Hemodilution reduced maximum clot firmness (MCF), whereas whole blood clotting time (CT) and maximum velocity remained unaffected. All coagulation factor activities were reduced. Fibrinogen, FII, FXIII and FX activities decreased significantly below the levels expected from dilution. The endogenous thrombin potential was unchanged. Ex vivo addition of fibrinogen normalized the reduced MCF and increased the maximum velocity, whereas PCC, rFVIIa and platelets shortened the CT but showed no effect on the reduced MCF. Conclusions: Acquired fibrinogen deficiency seems to be the leading determinant in dilutional coagulopathy, and ex vivo addition corrected the coagulopathy completely.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

A series of studies have documented that infusion of colloid plasma expanders induces coagulopathy and increases the risk of bleeding and transfusion requirements [1,2]. During the past decade, there has been considerable debate on the mechanisms involved in dilutional coagulopathy. Some reports have suggested that colloids and dextrans reduce the levels of von Willebrand factor (VWF) and factor VIII (FVIII) [3–5], whereas other studies have found masked expression of glycoprotein IIb/IIIa on activated platelets [6,7]. Previous in vitro studies from our laboratory have suggested that hydroxyethyl starch (HES)-induced dilutional coagulopathy is caused by acquired fibrinogen deficiency or dysfunction that can be completely corrected by substitution with a fibrinogen concentrate [1]. A series of other in vitro studies, animal studies, and retrospective clinical reports, as well as a few prospective studies, have supported the importance of fibrinogen deficiency and the efficacy of fibrinogen substitution therapy [8–13]. Historical data indicate that fibrinogen is the earliest coagulation factor to reach critically low threshold levels (< 1g L−1) in severely bleeding patients [14]. However, the critical level of fibrinogen has recently been subject to ongoing debate, and as a result the level of intervention has been set substantially higher at more centers. A recent study in obstetric patients showed that fibrinogen level was the main determinant of coagulopathy in postpartum hemorrhage [15], and another study in patients undergoing cardiac surgery also found fibrinogen to be the main predictor of postoperative bleeding [16]. In one laboratory study, thrombin generation was significantly reduced following in vitro dilution, most pronouncedly with HES as compared with crystalloids [17]. Another laboratory study showed that thrombin generation remained unchanged following dilution up to a level of 30% [18]. In conclusion, accumulating experimental evidence suggests that coagulopathy following moderate HES dilution (∼ 30%) is caused predominantly by fibrinogen deficiency and not abnormal thrombin generation. However, a recent animal study has suggested that the main issue of dilutional coagulopathy is a concerted reduction of fibrinogen, coagulation factor II (FII), FVII, FIX and FX activities, and substitution with prothrombin complex concentrate improved hemostatic efficacy [19]. Finally, a study found that thrombin generation was augmented by pharmacologic concentrations of activated recombinant FVIIa (rFVIIa), which seemed to improve coagulation parameters and support hemostasis [20]. So far, the mechanisms of dilutional coagulopathy have mainly been explored using in vitro human or animal studies. We recently conducted a prospective clinical trial in 20 patients undergoing elective cystectomy in which blood loss was substituted 1 : 1 with HES  130/0.4 until a level of dilution of 30% was reached [21]. The present article reports a detailed biochemical description of the mechanism of coagulopathy following systemic in vivo dilution with HES  130/0.4 (Voluven; Fresenius Kabi AB, Uppsala, Sweden) in bleeding patients. Furthermore, the article presents a systematic ex vivo test of various hemostatic interventions conducted on patients’ whole blood, including fresh frozen plasma, platelets, prothrombin complex concentrate (PCC), rFVIIIa, and FVIII.

The aims of the present study were to: (i) investigate changes in platelet count, thrombin generation, and dynamic whole blood clot formation, as well as the activities of VWF, FI, FII, FVII, FVIII, FIX, FX and FXIII, following 30%in vivo hemodilution with HES  130/0.4; and (ii) investigate the hemostatic effect of ex vivo administration of various hemostatic agents as listed above. We hypothesized that acquired deficiency of fibrinogen is the main determinant of dilutional coagulopathy.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Study subjects

The study was approved by the Danish Medicines Agency (EudraCT number 2007-00026-44), the Regional Biomedical Human Ethics Committee (reference code # 2007-0037), and the Danish Data Protection Agency (reference code # 2007-41-05849), and was conducted in accordance with the Note for Guidance on Good Clinical Practice (CPMP/ICH/135/95) and the Declaration of Helsinki. Patients older than 17 years of age who were admitted for elective radical cystectomy suffering from localized bladder cancer were considered to be candidates for inclusion in the study. The exclusion criteria were: (i) presence of coagulation disorders, defined as abnormal values of platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, antithrombin, or D-dimer; (ii) treatment with oral vitamin K antagonists; (iii) intake of non-steroidal anti-inflammatory drugs within 2 days prior to surgery; (iv) renal or hepatic dysfunction; (v) ischemic heart disease; (vi) pregnancy; and (vii) known hypersensitivity to HES.

Hemostatic agents for ex vivo investigation

The following hemostatic agents were investigated (values in the parentheses indicate the final concentration in the test cuvette whereas the corresponding clinical dose is entered in the brackets): rFVIIa [NovoSeven, 2 μg mL−1 (100 μg kg−1); NovoNordisk, Bagsvaerd, Denmark], fibrinogen concentrate [Haemocomplettan, 2.35 mg mL−1 (60 mg kg−1); CSL Behring, Marburg, Germany], PCCs [Beriplex, 0.25 IE mL−1 (17.5 IE kg−1); CSL Behring], and FXIII [Fibogammin, 0.25 IE mL−1 (17.5 IE kg−1); CSL Behring); fresh frozen plasma (5 mL kg−1) and platelet concentrate (5 mL kg−1) were in-house preparations. Plasma volume was estimated with the following formula: plasma volume in liters = (body weight in kilograms) × 0.045.

Anesthetic protocol and procedure of in vivo hemodilution

All patients were anesthetized and mechanically ventilated. Hypothermia was prevented by using a circulating warm air blanket as all infused fluids were preheated. From initiation of the surgical procedure, Ringer–acetate (Fresenius Kabi AB) was infused at 4 mL kg−1 h−1. The level of hematocrit was measured using a Radiometer ABL  625 analyzer (Radiometer Medical A/S, Copenhagen, Denmark). Lost blood was continuously substituted for 1 : 1 with HES  130/0.4 (Voluven; Fresenius Kabi AB).

Blood sampling and thromboelastometry whole blood coagulation analysis

Blood samples were obtained at baseline, prior to induction of surgery, and after 30%in vivo dilution with HES  130/0.4, defined as a 30% reduction in hematocrit level from baseline. Blood was drawn from the arterial catheter into VenoJect tubes (Terumo Europe, Leuven, Belgium; trisodium citrate, 0.129 m, 3.2% w/v), at a volume ratio of 1 : 10. To avoid artificial contact activation, blood sampling tubes for thromboelastometry were preincubated with corn trypsin inhibitor (Haemotologic Technologies Inc., Essex Junction, VT, USA) at a final concentration of 100 μg mL−1 [22]. Tubes for single coagulation factor measurement were centrifuged for 25 min at 2800 × g and 4 °C, and platelet-free plasma was frozen at − 80 °C. Dynamic whole blood coagulation profiles were recorded within 2 h using thromboelastometry (ROTEM Thromboelastometry; Pentapharm, Munich, Germany), as previously described [23]. In brief, ROTEM plastic cups prewarmed to 37 °C were loaded with 300 μL of whole blood and spiked with 20 μL of buffer (20 mm HEPES, 150 mm NaCl, pH 7.4) or 20 μL of the investigated hemostatic agent. The coagulation process was activated with tissue factor (Innovin; Dade Behring, Marburg, Germany) at a final dilution of 1 : 17 000 and recalcified with 0.2 m CaCl2. Thus, the final volume was 340 μL throughout. All analyses were processed in duplicate for a minimum of 45 min. Standard thromboelastometry parameters such as clotting time (CT) (s) and maximum clot firmness (MCF) (mm) were recorded. The digitalized raw signal was processed further using DyCoDerivAn software (AvordusoL, Risskov, Denmark) to obtain the dynamic velocity parameter maximum velocity (mm × 100 s−1) and time until maximum velocity, as previously reported [23].

Measurement of single factor levels and activity

Clotting activities of FII, FVII, FVIII, FIX and FX were determined by a one-stage method on Thrombolyzer Chrom Equipment (Benk Electronic, Norderstedt, Germany), using plasmas deficient in FII, FVII, FVIII, FIX and FX (Cryocheck, Dartmouth, Canada) as substrate, and recombinant human tissue factor (Innovin; Dade Behring) or Platelin  LS (Biomerieux, Herlev, Denmark) as the activating agent. FXIII transglutaminase activity was determined, using the Berichrom FXIII kit, on an automated coagulation analyzer (BCT, Dade Behring Coagulation Timer; Dade Behring).

Von Willebrand ristocetin cofactor activity was measured as agglutination of stabilized platelets contained in BC von Willebrand reagent (Dade Behring) together with ristocetin. All measurements were performed on a BCT Coagulation Analyzer (Dade Behring).

Levels of fibrinogen were measured according to the Clauss method, using the STAR Evolution analyzer (Diagnostica Stago (DS), Asnières, France) and thrombin reagent (STA-Fibrinogen; Diagnostica Stago) as activator. APTT and PT were assessed on the STAR Evolution analyzer (Diagnostica Stago), using commercially available reagents: APTT (phospholipids reagent, Platelin  LS; Organon, Munich, Germany), and PT (calcium–thromboplastin reagent, STA-Neoplastin; Diagnostica Stago). Antigen levels of fibrinogen were determined by means of nephelometry (BN ProSpec; Dade Behhring), using antiserum from rabbit against human fibrinogen (Dade Behring).

Thrombin generation

Thrombin generation was measured according to the protocol devised by Hemker [24], using a calibrated, automated thrombogram (Thrombinoscope BV, Maastricht, the Netherlands). Blood samples were centrifuged at 2800 × g for 25 min at 4 °C to obtain platelet-poor plasma. A 96-well microtiter plastic plate (Immulon  2HB clear 96-well; Thermo Electron Corporation, Vantaa, Finland) was prepared with 80 μL of platelet-poor plasma, followed by 20 μL of activator containing both a mixture of tissue factors (Innovin; Dade Behring) at a final dilution of 1 : 7000 and phospholipid-TGT (Rossix, Mölndal, Sweden) at a final concentration of 4 pm. After a brief incubation, 20 μL of thrombin substrate (Fluo-Substrate; Thrombinoscope) was added automatically. All reagents were prewarmed to 37 °C. Continuous development of thrombin was recorded on a Fluoroscan Ascent fluorimeter (Thermo Electron Corporation). The measurements were performed in triplicate, with each well calibrated to a parallel well with a thrombin calibrator (Thrombin calibrator TS  20.0; Thrombinoscope) with known thrombin-like activity.

Statistics

Statistical analysis was performed using the statistical software Graph Pad InStat (version 3.00; Graph Pad Software, San Diego, CA, USA). Groups were pretested for equal standard deviations using the method of Bartlett, and were estimated to follow a Gaussian distribution based on the methods of Kolmogorov and Smirnov. Effects of the hemostatic intervention and analysis of relative differences were tested using non-parametric Kruskal–Wallis test swith Dunn post hoc t-tests. Effect from haemodilution was tested using parametric paired t-test or Wilcoxon matched pairs test if samples were not from Gaussian distribution. A p-value less than 5% (P < 0.05) was considered statistically significant. Sample size of the clinical study was based on parameters presented elsewhere [21].

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Patient characteristics

A total of 20 patients were included in the study (two females and 18 males) with an average age of 63 years (range, 52–77 years) and a mean weight of 77 kg (range, 68–92 kg). All patients had preoperative levels of hemoglobin, platelets, APTT, PT, fibrinogen, antithrombin and D-dimer within the normal reference interval. Blood loss and fluid resuscitation during the operation resulted in a significantly decreased baseline hematocrit value, from 0.43 [standard deviation (SD), 0.028] to 0.29 (SD, 0.022), corresponding to a degree of dilution of 32%. After hemodilution, all of the standard parameters of coagulation decreased significantly, except for a non-significant increase in the levels of D-dimer (Table 1).

Table 1.   Laboratory characteristics before (baseline) and after 30%in vivo hemodilution with hydroxyethyl starch 130/0.4
 Baseline30% HemodilutionAbsolute decreaseRelative change (%)
  1. Data presented as mean ± standard deviation. Values in parentheses indicate normal reference ranges. APTT, activated partial thromboplastin time; VWF, von Willebrand factor. n = 20. *Significantly different from baseline value. Relative decrease significantly different from expected decrease (hemotocrit).

Hematocrit (0.28–0.47)0.43 ± 0.030.29 ± 0.02*0.14 ± 0.03− 32 ± 5
Standard coagulation parameters
 Platelet count, 109 L−1 (150–450)248 ± 66181 ± 50*61 ± 25− 27 ± 5
 APTT, s (25–38)32 ± 3.943 ± 11*9.2 ± 5+ 28 ± 13
 Prothrombin time, relative (> 0.80)0.93 ± 0.080.69 ± 0.08*0.25 ± 0.06− 27 ± 6
 Antithrombin, 103 IU L−1 (0.88–1.24)0.94 ± 0.080.56 ± 0.07*0.40 ± 0.06− 42 ± 5
 D-dimer, mg L−1 (< 0.50)0.54 ± 0.371.4 ± 2.60.75 ± 2.36+ 99 ± 177
Single coagulation factors
 Fibrinogen (μm)
  Ad modum Clauss9.5 ± 1.95.1 ± 0.8*4.04 ± 0.90− 44 ± 4.4
  Antigen level10.2 ± 1.95.9 ± 1.5*4.3 ± 0.90− 43 ± 6.1
 FII:C, U mL−1 (0.80–1.32)1.25 ± 0.20.68 ± 0.1*0.56 ± 0.16− 44 ± 6
 FVII:C, U mL−1 (0.68–1.69)1.07 ± 0.20.73 ± 0.2*0.33 ± 0.11− 31 ± 7
 FVIII:C, U mL−1 (0.66–1.55)1.45 ± 0.60.88 ± 0.4*0.61 ± 0.48− 39 ± 16
 FX:C, U mL−1 (0.74–1.52)1.19 ± 0.20.73 ± 0.2*0.46 ± 0.11− 39 ± 6
 FIX:C, U mL−1 (0.69–1.49)1.22 ± 0.260.89 ± 0.16*0.32 ± 0.14− 28 ± 6.6
 FXIII:C, U mL−1 (0.61–1.77)1.26 ± 0.20.71 ± 0.1*0.54 ± 0.12− 43 ± 6
VWF ristocetin cofactor, U mL−1 (0.47–1.59)1.51 ± 0.601.02 ± 0.38*0.49 ± 0.38− 30 ± 14
Endogenous thrombin potential (nm × min)1471 ± 3091532 ± 24737 ± 164 + 3.8 ± 11

Whole blood clot formation and thrombin generation potential – effect of in vivo hemodilution

In vivo hemodilution with HES  130/0.4 induced a coagulopathy characterized by significantly reduced MCF. CT, maximum velocity of clot formation and time to maximum velocity remained unaffected by hemodilution (Fig. 1).

image

Figure 1.  Parameters of thromboelastometric whole blood clot formation before (baseline) and after 30%in vivo hemodilution (squares) with hydroxyethyl starch 130/0.4 and after ex vivo (open circles) addition of fibrinogen, prothrombin complex concentrate (PCC), factor VIII (FVIII), fresh frozen plasma (FFP), activated recombinant FVII (rFVIIa), and platelets. n = 20. *Significantly different from baseline value. ¤Significantly different from 30% dilution. MaxVel, maximum velocity.

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Activities of FII, FVII, FVIII, FIX, FX, FXIII, and VWF – effect of in vivo hemodilution

Following in vivo hemodilution, there was a pronounced and significant reduction in the activities of FII, FVII, FVIII, FIX, FX, FXIII, and von Willebrand ristocetin cofactor. The activities of FII, FX, FXIII and fibrinogen were found to be decreased beyond the expected level after dilution (32%). No statistically significant difference was detected between antigen levels of fibrinogen and functional fibrinogen measurement using the Clauss method. In contrast, FIX was reduced less than expected (Table 1). There was no registered change in the endogenous thrombin potential following 32% hemodilution (Table 1).

Effect on whole blood clot formation of ex vivo addition of hemostatic agents

The CT was significantly shortened by ex vivo supplementation with PCC, rFVIIa, and platelets. Fibrinogen increased maximum velocity of clot formation, whereas time to maximum velocity was significantly shortened following in vitro addition of PCCs, rFVIIa, and platelets. Fibrinogen concentrate was the only reagent that significantly improved MCF.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

During the past decade, a series of reports have described the development of a coagulopathy following hemodilution with synthetic colloid plasma expanders, in particular when using HES  130/0.4. Various hypotheses have been suggested, including the development of acquired fibrinogen deficiency, acquired reduced levels of von Willebrand factor and FVIII, and as compromised expression of glycoprotein IIb/IIIa. Most studies addressing the mechanisms of dilutional coagulopathy have been based on laboratory in vitro experiments or animal models [1,10,11]. In contrast, the present study used blood samples from patients included in a systematic prospective clinical study, having blood loss meticulously substituted 1 : 1 with HES  130/0.4 until they reached a level of dilution of ∼ 30%. The present study verifies the development of a coagulopathy following in vivo hemodilution with HES  130/0.4, predominantly characterized by reduced MCF, the CT, maximum velocity and time to maximum velocity remaining unchanged. The MCF has been reported to primarily represent the level and function of platelets and levels of fibrinogen [25]. In addition, low levels of FXIII [26] or excessive fibrinolysis may be associated with reduced MCF [27]. Finally, inhibition of glycoprotein IIb/IIIa receptors diminishes the MCF [28]. In the present study, the most likely explanation for reduced MCF was reduced level of fibrinogen. Comprehensive measurement of hemostatic laboratory variables revealed that levels of platelets, fibrinogen, FII, FVII, FVIII, FIX, FX and FXIII were significantly reduced. Notably, following 30% hemodilution, levels of fibrinogen, FII, FX and FXIII were all decreased to values lower than expected from the dilutional effect. This observation could reflect excess loss, consumption, specific impairment, or assay discrepancy. Fibrinogen deficiency following HES dilution verifies several laboratory and animal studies. Specific impairment of FXIII transglutaminase activity on fibrin polymers has previously been described following colloid supplementation [29]. These observations are further supported by a recent study of Korte et al. [30], who reported not only a reduced decrease in MCF but also reduced blood loss in bleeding patients randomized to an FXIII concentrate. However, in the present study, ex vivo supplementation with FXIII did not improve the compromised MCF.

The low levels of FII and FX seem more likely to represent excess loss or consumption. However, the deficiency of FII and FX seems to be balanced by loss of anticoagulants, because the endogenous thrombin potential and CT, maximum velocity and time to maximum velocity remained unchanged following dilution. Our finding of uncompromised thrombin generation is supported by Nielsen [18], whereas other experimental and animal studies have reported a decrease in thrombin generation [17,31]. It may be speculated that compromised thrombin generation will emerge as a result of further decreases in the levels of prothrombin. Thus, Al Dieri et al. [32] investigated the relationship between clotting factor concentrations, parameters of thrombin generation and the amount of blood loss in patients with different congenital coagulation factor deficiencies; bleeding tendency was directly associated with the amount of thrombin generation, which varied linearly with the FII concentrations.

The platelet count decreased by ∼ 25% following hemodilution. Previous studies using whole blood thromboelastometry and activation with minute amounts of tissue factor have shown that the MCF and maximum velocity of whole blood clot formation do not reach abnormal levels before the platelet count reaches a threshold at 60 × 109 L−1 [33]. Thus, the observed decrease in MCF seems not to be caused by a reduction in the platelet count. Platelet aggregation was not assessed in the present study; however, other studies have indicated that rapidly degradable HES (such as HES  130/0.4) does not impair platelet function [34,35]. Finally, ex vivo addition of platelets only shortened the CT, the MCF remaining reduced. Patients undergoing surgery had a marginal, non-significant increase in D-dimer level following hemodilution. We did not investigate other markers of fibrinolysis; however, none of the thromboelastometry profiles showed signs of accelerated clot degradation. The reduced MCF was completely reversed following ex vivo addition of fibrinogen, as suggested in a series of other reports [1,10,11]. Finally, the present ex vivo findings are further supported by our parallel clinical study, showing significant hemostatic potential of in vivo supplementation with a fibrinogen concentrate [21].

Hemodilution of 32% with HES  130/0.4 prolonged the APTT and PT. This most likely reflects reduced levels of fibrinogen, FII, FVII, FVIII, FIX, and FX. In our study, PCC, rFVIIa and platelets all shortened CT and time until maximum velocity. This finding corresponds well with previous reports [19,36]; however, the clinical relevance may be questionable, because the coagulopathy following 30% hemodilution appears to be primarily defined by reduced MCF and not abnormal thrombin generation. It would have been expected that thrombin generation would be reduced following excessive hemodilution. Addition of fresh frozen plasma showed no hemostatic effect; however, the amount added was less than the clinically recommended supplementation.

Acquired von Willebrand syndrome and decreased levels of FVIII have been repeatedly observed [37,38] after hemodilution with slowly degradable HES, that is, high/medium molecular weight and high degree of hydroxyethyl motif substitution. However, so far, rapidly degradable HES has not been reported to reduce the levels of FVIII and VWF [39,40], and this is confirmed by the present study.

In vivo hemodilution with HES  130/0.4 reduced the MCF and induced a decrease in the levels of fibrinogen, prothrombin, FX and FXIII below the values expected from the degree of dilution. The endogenous thrombin potential was unchanged. Ex vivo addition of fibrinogen reversed the coagulopathy, whereas addition of PCC, rFVIIa and platelets reduced the CT to shorter than normal. In conclusion, following 30%in vivo hemodilution with HES  130/0.4, the most important mechanism of the coagulopathy appears to be a state of acquired fibrinogen deficiency.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

The authors are grateful for the excellent laboratory assistance from K. Christiansen and L. Norengaard. We also thank the staff at the Department of Urological Anaesthesiology and Department of Urology.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

The study was supported by an unrestricted research grant from CSL Behring (Marburg, Germany), the University of Aarhus Research Foundation, and the A. P. Møller and Hustru Chastine Mc-Kinney Møllers Foundation. None of the investigators received any personal honoraria for performing the study. Furthermore, the sponsors had no influence on the design of the study or on the analysis and interpretation of the results.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References