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CARDIOVASCULAR ANESTHESIA

The Effect of Fibrinogen Substitution on Reversal of Dilutional Coagulopathy: An In Vitro Model

Dietmar Fries^*, Petra Innerhofer^*, Christian Reif, Werner Streif, Anton Klingler, Wolfgang Schobersberger, Corinna Velik-Salchner^*, and Barbara Friesenecker^*

Departments of *Anaesthesiology and Critical Care Medicine and Pediatrics and Division of Theoretical Surgery, Innsbruck Medical University; and University for Health Science, Medical Informatics and Technology Tyrol (UMIT), Innsbruck, Austria

Address correspondence and reprint requests to Dietmar Fries, PhD, Department of Anaesthesiology and Critical Care Medicine, Innsbruck Medical University, Anichstr. 35, 6020 Innsbruck, Austria. Address e-mail to dietmar.fries{at}uibk.ac.at.

	Abstract

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Colloids and crystalloids are usually administered as treatment for hypovolemia in severely injured patients. However, dilution of clotting factors and platelets together with impaired fibrinogen polymerization are associated with fluid therapy and may aggravate hemorrhage, thus worsening final outcome of these patients. We investigated, in an in vitro model, whether the addition of fibrinogen to diluted blood samples can reverse dilutional coagulopathy. Blood from 5 healthy male volunteers was diluted by 60% using lactated Ringer's solution, 4% modified gelatin solution, or 6% hydroxyethyl starch 130/0.4, as well as the combination of lactated Ringer's solution with either of the 2 colloid solutions. Thereafter, aliquots of diluted blood samples were incubated with 3 different concentrations of fibrinogen (0.75, 1.5, and 3.0 mg/mL). Measurements were performed by modified thrombelastography (ROTEM®; Pentapharm, Munich, Germany). After 60% dilution, clotting times increased, whereas clot firmness and fibrin polymerization decreased significantly. After administration of fibrinogen, clotting times decreased and clot firmness, as well as fibrin polymerization, increased in all diluted blood samples. The effect of in vitro fibrinogen substitution on ROTEM® variables was dependent on the fibrinogen dosage and the type of solution used to dilute the blood samples.

	Introduction

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Trauma is the most frequent cause of death in Americans younger than of 34 yr, with up to 80% of all early deaths stemming from uncontrolled hemorrhage, 65% of which occur after hospital admission (1,2). Hemorrhage after trauma has two causes: mechanical damage and coagulopathy. Whereas mechanical damage may be surgically repaired, coagulopathy in the trauma patient is a more insidious process and a contributor to trauma mortality (3). The administration of fluids, and even red-cell concentrates, for managing massive blood loss causes clotting factor levels to decrease significantly through loss, consumption, and dilution, resulting in dilutional coagulopathy. Significantly decreased concentrations of fibrinogen seem to be the earliest occurring and most sensitive indicator of dilution and consumption, whereas critical levels of prothrombin, FV, FVII, FVIII, and platelets are reached in the late course of massive hemorrhage (4–8). In addition, even moderate dilution with colloids affects the coagulation system and, in particular, impairs fibrinogen polymerization, which finally results in decreased clot strength (9).

Polymerization of fibrinogen is the final common path of the hemostatic process after thrombin formation through activation of the plasmatic coagulation cascade. Low fibrinogen values have been shown to be associated with increased postoperative hemorrhage (10). Although fibrinogen concentration contributes substantially to final clot strength, few data are available to estimate the influence of this clotting factor on the reversal of dilutional coagulopathy. We demonstrate that substitution of fibrinogen partly reverses dilutional coagulopathy in an in vitro model.

	Methods

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Venous blood was withdrawn from 5 healthy male volunteers (mean age, 34.7 years) into citrate-containing tubes through a 1.2-gauge needle, thereby avoiding venous stasis. The volunteers showed normal renal and hepatic function and normal standard coagulation tests (plasma fibrinogen, 2.65 g/L [±0.28]; prothrombin time, 102% [±9.1]; partial thromboplastin time, 30 s [±3.5]; hemoglobin, 14.5 g/L [±0.79]; and platelet count, 253.8 x 10³/µL [±40.2]). None of the subjects received anticoagulant or antiplatelet medication during the last 2 wk before blood sampling. The study was approved by the Ethics Committee of the University of Innsbruck, and written informed consent was given by the volunteers.

Blood samples were analyzed using modified thrombelastography (ROTEM®; Pentapharm Co, Munich, Germany), which is based on the thrombelastography system (TEG®) of Hartert (11). Technical details of ROTEM® are described in the literature (12,13). ROTEM® shows good correlation with conventional TEG® analysis (14,15). Activation of the test samples accelerates measurements and enhances reproducibility as compared with conventional TEG® analysis (16). The variables of ROTEM® analysis are coagulation time (CT) corresponding to the reaction time (r time) of conventional TEG®, clot formation time (CFT) corresponding to the coagulation time (k time), anequivalent to the maximum amplitude (Fig. 1).

View larger version (26K): [in this window] [in a new window]   Figure 1. Thrombelastographic tracing showing the dynamics of development of the clot (coagulation time and clot formation time) and of clot firmness (maximum clot firmness).

Citrated blood was diluted by 60% with lactated Ringer's solution (RL) (Fresenius, Pharma Austria Co, Graz, Austria), 4% gelatin (Gelofusin®; B. Braun Co, Melsungen, Germany), or 6% hydroxyethyl starch (HES) 130/0.4 (Voluven®; Fresenius Co, Bad Homburg, Germany). The effect of the individual substances and that of the colloid and crystalloid solutions combined at a ratio of 1:1 (gelatin : RL, 6% HES 130/0.4 : RL) was evaluated. Afterwards, the diluted blood samples were incubated with 3 different concentrations of fibrinogen (0.75, 1.5, and 3.0 mg/mL) over 30 min. Transferring this in vitro model to an in vivo situation, fibrinogen concentrations corresponded approximately to an administration rate of 3–9 g of fibrinogen in an adult patient with approximately 70 kg of body weight. ROTEM® measurements were performed at 37°C in 300-µL undiluted (baseline) and diluted blood samples with and without the addition of fibrinogen. According to the manufacturer's instructions, samples were recalcified with 20 µL of CaCl₂ 0.2 M (Start-TEM®), activated with 20 µL of tissue thromboplastin (ExTEM®) alone and in the presence of cytochalasin D (FibTEM®), to determine the functional fibrinogen component of the formed clot (FibTEM®). All reagents were purchased from Nobis Co (Endingen, Germany). Data were collected for 30 min. The following variables were determined: CT (seconds), CFT (seconds), MCF (millimeters) and the angle for extrinsically activated tests without cytochalasin (ExTEM®), and MCF for tests containing cytochalasin (FibTEM®).

A repeated-measures analysis of variance was applied to analyze the influence of dilution and substitution of fibrinogen on thromboelastographic variables. Various fibrinogen concentrations were compared using an analysis of variance after subtracting the 60% dilution value and Tukey post hoc tests for individual intergroup comparisons. A P value less than 5% was considered statistically significant.

	Results

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As compared with baseline values, all ROTEM® variables were significantly impaired after 60% dilution with RL, gelatin, and 6% HES 130/0.4, as well as with the combination of the colloid and crystalloid solutions. This effect was significantly more pronounced in the blood samples diluted with 6% HES 130/0.4 and the combination of RL and 6% HES 130/0.4 (Fig. 2, C and E) than in the samples diluted with RL and gelatin individually or combined (Fig. 2A, B, and D).

View larger version (39K): [in this window] [in a new window]   Figure 2. Coagulation time (CT in seconds), clot formation time (CFT in seconds), maximum clot firmness (MCF in millimeters), and angle of extrinsically activated thrombelastographic measurements. Measurements were performed in undiluted blood (Baseline), after 60% dilution (Dilution) with lactate Ringer's solution (RL) (A), gelatin (B), 6% hydroxyethyl starch (HES) 130/0.4 (C), and the combination of RL with gelatin (D) or with 6%HES 130/0.4 (E) in a ratio of 1:1, as well as after substitution with 0.75 mg/mL (Fib-0.75), 1.5 mg/mL (Fib-1.5), or 3.0 mg/mL (Fib-3.0) of fibrinogen. *Statistically significant difference (P < 0.05) between baseline measurement versus diluted blood without and with additional fibrinogen.

After administration of fibrinogen, CT and CFT decreased and -angle, MCF, and fibrin polymerization (FibTEM®) increased in all diluted blood samples. The effect of fibrinogen substitution on ROTEM® variables was dependent on the fibrinogen dosage and the type of solution used to dilute the blood samples.

In the RL-diluted blood samples, administration of even the smallest concentration of fibrinogen decreased CT to a level comparable to that of the undiluted samples. CFT, MCF, and angle normalized after the administrations of 1.5 mg/mL and 3.0 mg/mL of fibrinogen. FibTEM® showed values similar to those in the undiluted samples, even after the addition of 0.75 mg/mL of fibrinogen. After the administration of larger doses of fibrinogen, FibTEM® measurements surpassed the results for the undiluted samples. However, MCF in the ExTEM® analysis did exceed baseline values (Fig. 2A).

Dilution with gelatin and the combination of RL and gelatin affected the results of the ROTEM® measurements more than dilution with RL alone. All variables, except ExTEM®-MCF, normalized after the administration of 1.5 mg/mL or 3.0 mg/mL of fibrinogen (Fig. 2, B and D).

In the blood samples diluted with 6% HES 130/0.4 or the combination of RL with 6% HES 130/0.4, ROTEM® variables improved after the administration of fibrinogen but did not reach baseline values after the administration of 3.0 mg/mL of fibrinogen (Fig. 2, C and E).

	Discussion

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Hemodilution using RL, gelatin, or 6% HES 130/0.4, as well as the combination of RL with gelatin or 6% HES 130/0.4, resulted in a dilutional coagulopathy. ROTEM® variables improved after the administration of fibrinogen. The degree of the effect was dependent on the fibrinogen dose and the type of solution used to dilute blood samples, after fibrinogen administration of ROTEM® variables of the blood samples diluted with RL or gelatin normalized (Fig. 2, A, B, and D). However, fibrinogen failed to completely reverse coagulation variables in the blood samples diluted with 6% HES 130/0.4 or the combination of RL and 6% HES 130/0.4 (Fig. 2, C and E).

In the case of massive blood loss, the administration of colloid and crystalloid solutions is crucial. A systematic review summarizing the results of randomized controlled trials on resuscitation with colloids versus crystalloids in critically ill patients reported an absolute increase of 4% in the risk of mortality after volume resuscitation with colloid solutions (17). Dilutional coagulopathy and other hemostasis impairment may have contributed to this finding. Whereas crystalloid fluid resuscitation predominates in the United States, the consensus in Europe is that a combination of both crystalloid and colloid infusion solutions is beneficial. Worsened bleeding ultimately increases mortality, which is probably not only the result of increased perfusion pressure after fluid therapy, but also the consequence of dilution of clotting factors and the direct impairment of the coagulation system by colloids.

In addition to a dilutional effect, gelatin preparations also exert specific effects on the coagulation system. Gelatin solutions disturb the reticular fibrin mesh and reduce functional clot quality, as measured by thrombelastography, clot weight, and scanning electron microscopy (18). HES is associated with an increased tendency to bleed, especially when using solutions with a high molecular weight and a high replacement degree, although recent data show that the molecular weight mainly determines duration of intravascular persistence but is not the determinative factor in compromising coagulation (19). HES solutions cause a von Willebrand factor (vWF) type 1-like syndrome characterized by diminished FVIII activity and diminished vWF plasma levels (20). In previous in vitro and in vivo studies, we were able to show that HES 130/0.4 and HES 200/0.5 also impairs fibrin polymerization to a greater degree than gelatin (9,21).

At this time, few data have been published on possible approaches reversing dilutional coagulopathy. In Central Europe, fibrinogen concentrate is available for immediate and efficient treatment of fibrinogen deficiency caused by dilutional and consumptional coagulopathy. The clinical experience with this approach is promising, although data justifying this approach are lacking (22). Fenger-Eriksen et al. (23) tested in vitro the effects of FVIII and fibrinogen and washed platelets on blood samples diluted with HES, dextran, or isotonic saline. Confirming our in vitro results, only fibrinogen improved impaired hemostasis after hemodilution. Unfortunately, the authors did not show the results of the undiluted control measurements. Hence, they can only assume that the administration of fibrinogen is able to restore hemostasis after hemodilution or even improve hemostasis after dilution to an unknown degree. Further, the effect of fibrinogen was not tested on gelatin (which is still an important solution in volume replacement therapy) and the effects of combined crystalloid-colloid dilution were also not investigated. In a porcine model, we were able to demonstrate that the administration of fibrinogen not only improved impaired hemostasis after hemodilution with gelatin, but also reduced blood loss, even in the case of uncontrolled hemorrhage after liver laceration (24).

Summarizing our data and those of others, it seems that fibrinogen plays a key role in massive blood loss and that fibrinogen deficiency is clinically more important than loss of other coagulation factors or platelets. Nevertheless, only few studies have addressed this topic.

Hiippala et al. (8) and Singbartl et al. (25) established in vivo and in a mathematical model that fibrinogen deficiency developed earlier than any other hemostatic abnormality. McLoughlin et al. (26) investigated the effect of normovolemic dilution in eight patients and in a porcine model. Even then, thrombocytopenia did not reach critical levels, whereas deficiency of clotting factors, including fibrinogen, was manifest.

Furthermore, the fibrinogen analysis should be interpreted with caution. In test samples from 1373 subjects, prothrombin time-derived plasma fibrinogen and fibrinogen, obtained by the Clauss method, differed from immunological assays. The prothrombin time-derived fibrinogen and the fibrinogen after the Clauss method showed significantly smaller mean values (27). Moreover, Hiippala (28) pointed out that synthetic colloids interfere with fibrinogen assays. The samples diluted with colloids showed larger fibrinogen values than the samples diluted with saline. Thus, in the case of major bleeding with dilution and consumption of coagulation factors, and after the administration of large amounts of colloids, the real functional plasma fibrinogen value may be over-estimated by standard laboratory tests. In this situation, modified thrombelastography may be useful for promptly and accurately analyzing the functional fibrinogen actually available for polymerization (29).

In conclusion, we show that fibrinogen improves coagulation after in vitro hemodilution with crystalloids and colloids. Further animal and clinical studies are required to confirm our hypothesis that the in vivo administration of fibrinogen is an immediately possible and useful first step toward reversing dilutional coagulopathy, thereby reducing total blood loss and further transfusion requirement.

	Footnotes

 
Accepted for publication September 13, 2005.

	References

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Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999