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CARDIOVASCULAR ANESTHESIOLOGY

The In Vitro Effects of Fibrinogen Concentrate, Factor XIII and Fresh Frozen Plasma on Impaired Clot Formation After 60% Dilution

Thorsten Haas, MD^*, Dietmar Fries, MD, Corinna Velik-Salchner, MD^*, Christian Reif, Anton Klingler, MD, PhD, and Petra Innerhofer, MD^*

From the Departments of *Anaesthesiology and Critical Care Medicine, General and Surgical Critical Care Medicine, Pediatrics, General and Transplant Surgery, Division of Theoretical Surgery, Innsbruck Medical University, Innsbruck, Austria.

Address correspondence and reprint requests to Thorsten Haas, MD, Department of Anaesthesiology and Critical Care Medicine, Innsbruck Medical University, Anichstrasse 35, 6020 Innsbruck, Austria. Address e-mail to thorsten.haas{at}i-med.ac.at.

Abstract

BACKGROUND: Previous investigations have shown that increasing fibrinogen concentration improves dilution-dependent impairment of clot formation. We conducted an in vitro study to explore whether substitution with fibrin-stabilizing factor XIII (FXIII) combined with fibrinogen promotes further improvement of clot formation, and whether fibrinogen administration as concentrate or fresh frozen plasma (FFP) results in comparable effects.

METHODS: Blood from six healthy donors was diluted by 60% using lactated Ringer’s solution. Aliquots of diluted blood samples were incubated with two different doses of fibrinogen concentrate, FXIII concentrate, the combination of both, or with two different doses of FFP. Using thrombelastometry (ROTEM®) blood samples were analyzed at baseline (undiluted), after dilution and after supplementation. Variables were analyzed for changes from baseline, and effects of fibrinogen concentrate alone or combined with FXIII were compared with effects observed with corresponding FFP doses.

RESULTS: After 60% in vitro dilution of blood all ROTEM parameters and global coagulation tests changed significantly. Among the substitutes tested FXIII alone had no effect, the combination with fibrinogen improved coagulation time, angle and fibrinogen/fibrin polymerization significantly more than did small-dose fibrinogen alone. After substituting fibrinogen, median values of all ROTEM variables were within the normal range, thereby showing dose dependency but also significant differences (P = 0.027) from corresponding FFP doses (EXTEM MCF FFP small dose [38 (35, 40.3) mm)], which enabled only coagulation time to be shortened to baseline levels.

CONCLUSIONS: Supplementation of fibrinogen restored all ROTEM parameters after dilution. This effect was partially enhanced by adding FXIII and was significantly stronger than for FFP substitution.

Administration of crystalloid or colloid fluids is usually recommended in surgical and trauma patients to maintain normovolemia. However, all fluids cause dilutional coagulopathy and exert specific effects on the clotting process. Using thrombelastographic techniques, which display the speed and quality of initiation of coagulation, and also clot formation, several in vitro, experimental and clinical studies have shown that dilution slows clot propagation and diminishes clot strength because it impairs fibrinogen/fibrin polymerization. As the degree of dilution increases, the reduction in clot firmness is also accompanied by impaired initiation of coagulation,1,2 which is observed when coagulation factor levels decrease to <20%–30%.3 Factor XIII (FXIII) is a transglutaminase that, in the presence of fibrin, greatly accelerates the activation process by cross-linking fibrin polymers and thus influencing formation of a stable clot.4 Clot quality is further promoted by increasing the clot’s resistance to fibrinolysis through FXIIIa-mediated covalent binding of ₂ plasmin inhibitor to fibrin molecules.5

In clinical practice, and following the recommendations of official societies, coagulopathy is usually treated by administering fresh frozen plasma (FFP), cryoprecipitate, or platelets.6 However, neither transfusion of FFP or platelets, nor administration of cryoprecipitate has been sufficiently investigated for treatment of intraoperative coagulopathy, and at 10–15 mL/kg, the recommended dosage of FFP has been questioned as being ineffective.7 Furthermore, cryoprecipitate is not available in most European countries, and transfusion of FFP or platelets should be restricted for several reasons including transmission of viral infection and potential bacterial contamination. Additionally, FFP in particular has been shown to provoke transfusion-related acute lung injury.8 Nevertheless, administration of purified fibrinogen concentrate in vitro, in an animal and clinical studies and was able to improve dilution-dependent impaired clot formation,9–12 whereas the effects of neither factor FXIII substitution nor FFP administration have been adequately investigated in this context. Although fibrinogen and FXIII contribute substantially to clot strength, no data are available to estimate the influence of the combination of these clotting factors toward improving clot firmness. Moreover, the effect of these clotting factor concentrates has never been compared with that of FFP, which is the standard of care for treatment of dilutional coagulopathy.

Therefore, we conducted this in vitro study to compare the effects of fibrinogen concentrate, FXIII, fibrinogen and FXIII, or FFP on coagulation kinetics in whole blood diluted by 60% with lactated Ringer’s solution (RL). We decided to induce dilution-dependent impairment of the clotting process with 60% dilution to observe the clinically relevant reduced clot firmness and prolonged initiation of coagulation that are to be expected during profound dilution only. Moreover, especially when hemostatic competence is severely compromised, as in trauma patients, effective substitution is mandatory to reduce further blood loss.

METHODS

The study was approved by the Ethics Committee of Innsbruck Medical University. After written informed consent was obtained, venous blood was withdrawn from 6 healthy male blood donors into citrated sample tubes through a 1.2-gauge needle, thereby avoiding venous stasis. The volunteers showed normal coagulation tests, had no history of coagulopathy, and none had received anticoagulant or antiplatelet medication during the last 2 wk before blood sampling.

Citrated blood was diluted by 60% with RL (Fresenius, Pharma Austria Co, Graz, Austria). ROTEM® analyses were performed in undiluted and diluted blood samples and after adding fibrinogen concentrate (Hemocomplettan P®, CSL Behring, Marburg, Germany), FXIII concentrate (Fibrogammin P®, CSL Behring), fibrinogen with FXIII, or FFP. Aliquots of 2 mL diluted blood samples were incubated with either 80 or 160 µL fibrinogen concentrate (small-dose and large-dose fibrinogen) corresponding to about 4 g (60 mg/kg) or 8 g (120 mg/kg) in a patient of 70 kg bodyweight or with 400 µL (or 800 µL) FFP corresponding to 15 mL/kg (or 30 mL/kg) in a patient of 70 kg bodyweight, or with 80 µL FXIII concentrate in 2.5 mL diluted blood corresponding to 5000 IU in a patient of 70 kg bodyweight, as well as with the combination of fibrinogen and FXIII (fibrinogen + FXIII). The described fibrinogen dosages were used because they refer to the fibrinogen content delivered by 1000 or 2000 mL FFP (4 g/L) at the recommended and probably more effective doses of 15 mL/kg, 30 mL/kg, respectively. Fibrinogen concentrate was diluted in 50 mL distilled water as usual in clinical practice to avoid the influence of altered viscosity. The volume of substitutes added differed, because it was our intention to reflect the additional dilution occurring in vivo (for small-dose FFP and fibrinogen 1000 mL vs 200 mL). Thus, the volume of substitute added was four-fold higher for FFP than for fibrinogen concentrate. The FXIII dosage was calculated to compensate for the decrease in FXIII activity resulting from 60% dilution, and the administered volume refers to that occurring in vivo after administration of 5000 IU FXIII concentrate in 40 mL.

All blood samples (undiluted, diluted, and after substitution) were analyzed at 37°C using thrombelastometry (ROTEM, Pentapharm, Munich, Germany), which is based on the thrombelastography system (TEG®) after Hartert. Technical details and clinical experience with TEG/ROTEM are reviewed in the literature.13 According to the manufacturer’s instructions, citrated blood (300 µL) was recalcified with 20 µL of CaCl₂ 0.2M (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. Cytochalasin D inhibits platelet degranulation and reorganization of the platelet’s cytoskeleton and prevents fibrinogen from binding to GPIIb/IIIa receptors. Data were collected for 30 min. In the present study, tissue factor-activated ROTEM tracings were analyzed for coagulation time, clot formation time (CFT), clot propagation ( angle), maximum clot firmness (MCF), and firmness of the fibrinogen/fibrin part of the clot (FibTEM MCF).

Additionally, in undiluted and diluted blood samples hematocrit, platelet count (platelet), prothrombin time, activated partial thromboplastin time, plasma fibrinogen concentration (fibrinogen), FXIII concentration, and antithrombin were determined by standard laboratory methods using the appropriate tests from Dade Behring, and the Amelung Coagulometer, Baxter, UK.

Sample size was calculated with nQuery Advisor® software (Statistical Solutions, Ireland) with a power of 90% and a two-sided of 5%, and was based on pilot data. A sample size of six blood samples per group allowed a difference of 14.5 mm (sd = 4.1) in MCF to be detected between substitution groups. If overall Friedman ANOVA was positive, a paired Wilcoxon Test was applied to analyze changes from baseline within substitution groups and to compare changes from baseline between substitution groups (small-dose and large-dose fibrinogen and corresponding FFP doses, fibrinogen alone and combined with FXIII). A P value of <0.05 was considered significant.

RESULTS

At baseline all ROTEM and standard laboratory variables were comparable among blood donors and within the normal range. When compared with baseline values, all standard laboratory (Table 1) and ROTEM parameters (Figs. 1A–E) changed significantly after 60% dilution with RL, but ROTEM parameters improved differently with the various substitutes.

View larger version (19K): [in this window] [in a new window]   Figure 1. A–E, Extrinsically activated measurements of coagulation time (CT in seconds), clot formation time (CFT in seconds), angle (in degrees), maximum clot firmness (MCF in millimeter), and fibrin polymerization (FibTEM MCF in millimeter). Measurements were performed in undiluted blood (baseline), after 60% dilution (dilution) with lactated Ringer’s solution, as well as after substitution with 80 µL (or 160 µL) fibrinogen concentrate (fibrinogen) in 2 mL diluted blood corresponding to 4 g (or 8 g) in a patient of 70 kg bodyweight, 80 µL factor XIII concentrate (FXIII) in 2.5 mL diluted blood corresponding to 5000 IU in a patient of 70 kg bodyweight, the combination of fibrinogen and FXIII (fibrinogen + FXIII), and 400 µL (or 800 µL) FFP in 2 mL diluted blood corresponding to 15 mL/kg (or 30 mL/kg) in a patient of 70 kg bodyweight. *P < 0.05 vs baseline, P < 0.05 small-dose fibrinogen vs small-dose FFP, P < 0.05 small-dose fibrinogen vs large-dose fibrinogen, OP < 0.05 small-dose fibrinogen vs fibrinogen + FXIII, P < 0.05 small-dose FFP vs large-dose FFP, =P < 0.05 large-dose fibrinogen vs large-dose FFP.

Prolonged initiation of coagulation, measured as coagulation time (Fig. 1A) shortened by all substitutes except FXIII alone, achieved times shorter than at baseline after adding fibrinogen and FXIII, or large-dose fibrinogen alone. Effects after small- and large-dose FFP were similar and not significantly different from those observed after administration of fibrinogen.

Speed of clot formation after initiation of coagulation is measured by CFT (Fig. 1B) and angle. CFT remained prolonged with all substitutes except large-dose fibrinogen. Substituting fibrinogen shortened CFT significantly more than did FFP at low (P = 0.028) and high dosages (P = 0.028). Increasing the FFP dosage had no effect, and the effectiveness of FXIII combined with fibrinogen was similar to that of fibrinogen alone.

Measurements of angle (Fig. 1C) reached and exceeded baseline values for fibrinogen concentrate combined with FXIII and for large-dose fibrinogen. Small- and large-dose fibrinogen administration restored angle significantly more than did corresponding FFP dosages (P = 0.026 and 0.027). Increase in angle was significantly greater with large-dose FFP than with small-dose FFP.

Clot firmness (Fig. 1D) remained below baseline for all substitutes except large-dose fibrinogen. No effect was registered for FXIII alone or combined with fibrinogen. Fibrinogen substitution improved clot firmness at low (P = 0.027) and high dosage (P = 0.026) significantly more than did corresponding small- and large-doses of FFP, and large-dose FFP substitution increased clot firmness significantly more than did small-dose FFP substitution (P = 0.042).

Fibrinogen/fibrin polymerization (Fig. 1E) improved dose-dependently to and above baseline levels with small- and large-dose fibrinogen alone and significantly more than with corresponding FFP substitution, which remained significantly different and below baseline with small-dose FFP. The combination of small-dose fibrinogen and FXIII increased fibrinogen/ fibrin polymerization significantly more than did small-dose fibrinogen alone (P = 0.034).

DISCUSSION

The current profound dilution of venous blood by 60% resulted in clinically relevant impairment of all ROTEM variables and standard coagulation tests, thereby reaching values deemed to require FFP, whereas thresholds for platelet administration were not reached. Guidelines suggest that prolongation of standard coagulation tests by >1.5-fold the normal values indicates a deficiency of the coagulation factors needed for sufficient thrombin generation and6,14 usually recommend the administration of 10–15 mL/kg FFP, although the efficacy of such single doses has been shown to be poor.7,15 Our in vitro model showed that all substitutes except FXIII alone were able to correct initiation of coagulation to baseline values. However, improvement of the subsequently occurring propagation of coagulation, clot firmness and fibrinogen/fibrin polymerization showed differences among the various substitutes. Median values of all variables of clot propagation and of clot quality improved to baseline values, and even above, in a dose-dependent fashion with fibrinogen addition, and significantly more than after corresponding addition of FFP. Significant differences for small- and large-dose FFP were observed only for changes in angle and clot firmness, which, however, remained below normal ranges, even after large-dose FFP administration.

These effects of fibrinogen administration confirm our earlier findings10 and those of Fenger-Eriksen et al., who additionally demonstrated that substitution with neither factor VIII nor platelets was able to produce comparable effects.11 When using pooled diluted plasma and incubation with fibrinogen and thus measuring fibrinogen/fibrin polymerization,12 Nielsen, too, found that fibrinogen was able to improve propagation and firmness of the fibrinogen/ fibrin part of the clot towards baseline in samples diluted with 0.9% NaCl (40%). The quality of fibrinogen/ fibrin polymerization depends not only on sufficient thrombin generation and fibrinogen concentration but also on the activity of FXIII. Severe hereditary deficiency of FXIII can cause life-threatening hemorrhage,16 and acquired FXIII deficiency has been identified as a cause of unexplained intraoperative bleeding17 associated with increased blood loss after cardiac surgery18,19 and increased rebleeding and the need for surgical revision after intracranial surgery.20 Although FXIII deficiency is presumed to develop at late stages of blood loss,21 it contributes substantially to clot strength4 and might be of greater importance than has so far been known. When using classical TEG and FXIII-deficient plasma in which all other coagulation factors remained normal, Nielsen et al.22 also showed that a significant decrease in fibrinogen/fibrin clot strength and angle is detectable when FXIII decreases below 60% and that initiation of coagulation is prolonged with further reduction of FXIII concentration <20%. In our study, FXIII activity decreased to median levels of 33% after dilution and was accompanied by deficiencies in the other factors influencing clot strength, namely fibrinogen concentration and platelet count, which might alter critically functional thresholds of FXIII. In this situation substituting FXIII alone did not show any detectable effect, but its combination with small-dose fibrinogen enhanced fibrinogen’s effect on angle and fibrinogen/fibrin polymerization. Interestingly, the combination of FXIII and fibrinogen produced significantly higher FibTEM levels than did fibrinogen alone, whereas EXTEM MCF readings, not influenced by cytochalsin D, did not. This might have been due to the fact that cytochalasin D inhibits both degranulation and GPIIb/ IIIa assembly by preventing intracellular microtubular formation, and thus prevents the release of platelet FXIIIa stores. Nevertheless, the role of intracellular FXIII has remained mostly unexplored.

Although the literature provides little evidence to substantiate the efficacy of FFP in reducing blood loss and improving coagulopathy,15 FFP is the routine treatment option for correcting intraoperative coagulopathy. Our results further support the results of others, in that we observed poor efficacy after FFP administration.7 It must be considered that the volumes of the tested substitutes (fibrinogen or FFP) were comparable in fibrinogen content, although the added volume differed; the largest amount was needed for FFP substitution, which consequently produced disproportionately greater dilutional effects. This also applies to the in vivo condition when FFP is administered or a comparable fibrinogen dosage is given as fibrinogen concentrate. Since FFP also contains considerable amounts of albumin and other proteins exhibiting osmotic effects, the resulting in vivo dilutional effect might be even more pronounced.23

Some limitations of our study need to be mentioned. First, we performed an in vitro study that lacked the contribution of endothelial and vascular factors to the clotting process but enabled standardization of other influencing variables. For functional analysis of hemostatic competence, we performed ROTEM analysis because there is growing evidence that thrombelastographic techniques (ROTEM/TEG) are superior to time-consuming standard laboratory tests in guiding intraoperative coagulation management.14,21 Nevertheless, transfusion algorithms rely on measuring fibrinogen concentrations. Data from two other studies in children and adults conducted by our study group24,25 show a strong and significant correlation comparable to the results observed in vitro by Nielsen et al.26 The question may arise whether clot size can be safely increased by supplementing fibrinogen without triggering thromboses. It is true that at levels far above 400 mg/dL fibrinogen might be associated with myocardial infarction.27 However, in cases with diagnosed impairment of fibrinogen polymerization, as observed in the present study, even the highest dosage used produced values within the normal range of 9–25 mm.25

In conclusion, our in vitro data show that all parameters of clot formation recovered dose-dependently after fibrinogen substitution, while substitution with FXIII alone, or even with FFP, was nearly ineffective. The higher dose of FFP improved clot strength significantly more, but was still not able to restore values to reference ranges. In cases showing poor improvement of angle and fibrinogen/fibrin polymerization after fibrinogen administration, a critical reduction in FXIII should be considered and possibly corrected by substituting FXIII, which might reduce the need for further fibrinogen administration.

Footnotes

Accepted for publication December 19, 2007.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Haas, T. Articles by Innerhofer, P. Search for Related Content PubMed PubMed Citation Articles by Haas, T. Articles by Innerhofer, P. Related Collections Cardiovascular Blood Coagulation