Fibrin patch in a pig model with blunt liver injury under severe hypothermia

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Fibrin patch in a pig model with blunt liver injury under severe hypothermia Christian Zentai, MD,a,b,* Till Braunschweig, MD,c Rolf Rossaint, MD,a Moritz Daniels, MD,a Michael Czaplik, MD,a Rene Tolba, MD,b and Oliver Grottke, MD, PhDa a

Department of Anesthesiology, RWTH Aachen University Hospital, Aachen, Germany Institute for Laboratory Animal Science, RWTH Aachen University Hospital, Aachen, Germany c Department of Pathology, RWTH Aachen University Hospital, Aachen, Germany b

article info

abstract

Article history:

Background: Rapid control of hemorrhage is one of the key aspects in trauma handling. To

Received 2 September 2013

cope with bleeding, local hemostatic approaches are useful, along with surgical and sys-

Received in revised form

temic homostatic therapy. In this experimental study, we investigated the efficacy of a

5 November 2013

fibrinogen/thrombin containing collagen patch (TachoSil) in a coagulopathic pig model

Accepted 7 November 2013

with blunt liver trauma under severe hypothermia.

Available online 15 November 2013

Methods: Eighteen anesthetized pigs underwent hemodilution by exchanging 70% of the blood volume with Ringer Lactate solution and hydroxyethyl starch 130/0.4 (1:1). Ten minutes

Keywords:

after induction of a grade III blunt liver trauma, the animals randomly received treatment

Coagulopathy

with TachoSil (FT-patch, n ¼ 9) or a collagen patch (Tachotop, control group, n ¼ 9). Blood loss,

Hemorrhage

hemodynamics, and coagulation parameters were observed for 2 h. To confirm the consis-

Fibrin sealant

tency of liver trauma, pathologic examination of the liver tissue was performed.

Liver injury

Results: Hypothermia (33.5 C  0.5 C) and hemodilution led to severe coagulopathy as

Hypothermia

measured by thromboelastometry and coagulation parameters. After trauma and patch

Pig

application, thromboelastometry and coagulation parameters in the control group showed

Trauma

further deterioration compared with the stable parameters in the FT-patch group. The total

Thromboelastometry

blood loss was significantly reduced in the FT-patch group (FT-patch: 1195 mL; control

Damage control surgery

group: 2495 mL; P < 0.001). Concordantly, the control animals were hemodynamically jeopardized to a higher degree. Microscopy confirmed a similar degree of liver injury. Conclusions: Despite severe hypothermia and coagulopathy, TachoSil provided effective hemorrhage control in pigs with blunt liver injury. Therefore, TachoSil demonstrated usefulness as an additional early therapy in cases of uncontrolled bleeding following severe trauma. ª 2014 Elsevier Inc. All rights reserved.

1.

Introduction

Trauma is an international cause of concern to the public. It is the leading cause of death for people aged 1e44 years in the

United States [1]. Hemorrhage is second on the list of early overall cause of trauma-related death [2]. Above all, it is the leading cause of preventable deaths [3]. In the previous decades, the mortality caused by exsanguination was reduced by

This study was performed at the RWTH Aachen University Hospital, Pauwelsstr. 30, D-52074 Aachen, Germany. * Corresponding author. Department of Anesthesiology, RWTH Aachen University Hospital, Pauwelsstr. 30, 52074 Aachen, Germany. Tel.: þ49 241 8035596; fax: þ49 241 8082406. E-mail address: [email protected] (C. Zentai). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2013.11.007

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the broader availability of better diagnostics (e.g., multi-slice CT), the availability of international guidelines [4], the implementation of viable trauma care algorithms (e.g., PreHospital Trauma Life Support and Advanced Trauma Life Support) [2], and the stringent pursuit of strategies such as damage control surgery and damage control resuscitation [5,6]. Damage control surgery (DCS) emphasizes the need to control bleeding and reduce contamination by limited surgery, followed by recovery and stabilization of blood parameters (including coagulopathy) before definitive surgical care [7e9]. However, DCS and the coherent surgical control of bleeding are often hampered by impaired hemostasis. Acute traumatic coagulopathy appears early after injury [10], is linked with a 4- to 5-fold increased mortality [11e13], and is detectable in approximately one-fourth to one-third of trauma patients at the time of presentation to the hospital [11,12,14]. Therefore, coagulopathy is often established at an early stage of trauma care and DCS. In combination with acute traumatic coagulopathy, it is an even greater challenge to achieve the primary goal of DCS, that is, control of bleeding, especially if noncompressible internal sources such as vessels or organs are accountable for the sustained hemorrhage. Hemostatic dressings directly applied to the bleeding area can provide local hemostasis and gain valuable time in situations of uncontrolled severe bleeding or even terminate bleeding. One of the commercially available carrier-bound fibrin sealants is TachoSil (Nycomed, Roskilde, Denmark), which is a ready-to-use patch of equine collagen coated with human fibrinogen and thrombin [15]. Both coagulation factors initiate the final steps of fibrin clot formation and are activated by contact with fluid and facilitate local hemostasis in the inflicted area covered by the patch. The efficacy of TachoSil in controlling bleeding has been evaluated in different surgical disciplines [16,17]. Overall, these studies demonstrate the positive impact of TachoSil on bleeding control. However, these trials did not investigate the efficacy of TachoSil under coagulopathic conditions and hypothermia. Using a coagulopathic pig model, we showed in a previously conducted experimental study that TachoSil effectively controlled bleeding and reduced mortality caused by exsanguination after blunt liver trauma under normothermia [18]. However, the majority of severely injured patients are hypothermic at admission, and hypothermia substantially contributes to coagulopathy [19e21]. Therefore, the present study evaluated the efficacy of TachoSil in a coagulopathic pig model under severe hypothermia with blunt liver injury. The primary objective was to determine the effectiveness of TachoSil in the control of bleeding and assess the impact on coagulation in a clinically relevant large animal model.

2.

Materials and methods

2.1.

Ethics and animals

This study was performed at the RWTH Aachen University Hospital (Aachen, Germany) according to German Animal Welfare law and following the Guide for the Care and Use of Laboratory Animals [22]. Official permission was granted by the appropriate governmental animal care and use office

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(Landesamt fu¨r Natur, Umwelt und Verbraucherschutz, Recklinghausen; No. 87-51.04.2010A300). Eighteen healthy male German landrace pigs (body weight 30e37 kg) obtained from a disease-free breeding farm were allowed to acclimatize in ventilated rooms for at least 5 days before surgery and were fasted the night before surgery with free access to water.

2.2.

Anesthesia

Premedication was administered by an intramuscular injection of 4 mg/kg azaperone (Stresnil; Janssen, Neuss, Germany) and 0.1 mg/kg atropine (Atropinsulfate; B. Braun, Melsungen, Germany). Anesthesia was induced by intravenous injection of 3 mg/kg propofol (Disoprivan; AstraZeneca, Wedel, Germany) via an ear vein for orotracheal intubation. Adequate anesthesia was maintained during the whole experiment by continuous infusion of fentanyl (Fentanyl-Rotexmedica; Rotexmedica, Trittau, Germany) at a concentration of 3e4 mg/ (kg$h) and inhalation of isoflurane (Forane; Abbott, Wiesbaden, Germany) at an end-tidal concentration of 1.2%. Animals were ventilated with 100% oxygen in a closed circuit (PhysioFlex; Dra¨ger, Lu¨beck, Germany) with 16e20 breaths/min and a tidal volume of 10 mL/kg to keep the end-tidal partial carbon dioxide concentration between 36 and 42 mmHg. To conform to basic fluid requirements, Ringer Lactated solution (RingerLactat-Lo¨sung [RL]; Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany) was infused continuously at a rate of 4 mL/(kg$h) before and a rate of 10 mL/(kg$h) after laparotomy. The animal blood temperature (measured constantly using a pulmonary artery catheter) was maintained between 36.5 C and 37.0 C with a warming pad until hemodilution. Continuous monitoring included electrocardiogram, tail pulse oximetry, blood temperature and cardiac output (CO), arterial and central venous pressures as well as pulmonary pressure using a standard anesthesia monitor (AS/3; Datex Ohmeda, Helsinki, Finland).

2.3. Surgical preparation, hemodilution, and induction of hypothermia Vascular access was secured by percutaneous insertion of two 8.5F catheters (for hemodilution: blood withdrawal and fluid resuscitation) in the left and right femoral veins. An 18G catheter was inserted into the femoral artery to monitor arterial blood pressure and collect blood for sampling. A third 8.5F catheter was surgically implanted in the right jugular vein for the placement of a pulmonary artery catheter in wedge position. After line placement, followed by a midline laparotomy with cystostomy and splenectomy (to avoid splenic blood pooling), baseline parameters for hemodynamics and laboratory tests were taken. A bolus of RL (37 C, three times the weight of the spleen) was administered to counteract the blood loss associated with the removal of the spleen. To provoke dilutional coagulopathy, approximately 70% of the pig’s estimated total blood volume [23] was withdrawn at a rate of 100 mL/min. The blood was collected and processed for later retransfusion using a cell saver system (Cell Saver 5; Haemonetics, Munich, Germany) with sodium citrate. The blood loss was substituted by RL and hydroxyethyl starch 130/

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0.4 (Voluven; Fresenius, Bad Homburg, Germany) in a ratio of 1:1. This fluid resuscitation was started once the mean arterial pressure (MAP) declined to a 40%e50% threshold of the baseline value. After fluid resuscitation, salvaged erythrocytes were retransfused to all animals (20 mL/kg within 10 min) to avoid early death from anemia. Severe hypothermia was induced reliably by the infusion of resuscitation volume at room temperature (approx. 20 C) and by uncovering the animals without active cooling. A further decrease of the animal’s core temperature was prevented by the intermittent or continuous use of a warming blanket (Warm Touch; Mallinckrodt Medical, Inc, Cd. Juarez, Mexico), stabilizing the temperature at 33 Ce34 C.

(MEK-6108; Nihon Kohden, Tokyo, Japan) calculated the platelet count and hemoglobin concentration (Hb). A ROTEM 0.5 (ROTEM; TEM International, Munich, Germany) was used for thromboelastometry (ExTEM assay) to measure the clotting time (CT), clot formation time (CFT), and maximum clot firmness (MCF). The CT was defined as the duration until clotting was primarily evident (standardized as amplitude of 2 mm); the CFT was defined as the length of time required for the amplitude to reach 20 mm and the MCF was depicted by the maximum amplitude and, therefore, reflected the strength of the resulting clot [30]. All coagulation samples were measured in duplicates.

2.6. 2.4.

Experimental protocol and liver injury

A comprehensible grade III blunt liver injury [24] was inflicted by manually squeezing the parenchyma of the left liver lobe placed between the branches of a custom-made clamp as described previously [18,25e29] with a digitally visualized and recorded force of 239e275 N (LabView 2011; National Instruments, Austin, TX). After a posttraumatic shock phase of 5 min, all the animals received a bolus of RL (6 mL/(kg$min) over 5 min) followed by a continuous infusion at a rate of 35 mL/ (kg$h). During these first 10 min, free bleeding was allowed. The resulting blood loss was determined by suctioning all the blood out of the abdominal cavity. Subsequently, animals were randomly assigned via the sealed envelopes method to either the FT-patch group (FT-patch, n ¼ 9; TachoSil; Nycomed) or the control group (Control, n ¼ 9; Tachotop; Nycomed). Both patches were placed covering the complete area of liver injury and were manually compressed for 3 min according to the manufacturer’s instructions [15] after short humidification with normal saline solution. The abdomens of all animals were coarsely closed with clamps for the rest of the observation phase that ended 120 min after liver injury. Animals surviving the observation period were euthanized with an overdose of fentanyl, propofol, and potassium chloride. After death, the abdomen was instantly reopened and the peritoneal cavity cleared again of all blood to determine the secondary and total blood loss. Postmortem, the inflicted liver lobe and samples from three different representative locations of the surrounding liver were obtained and fixed in 10% buffered formalin for pathologic examination.

The liver was removed shortly after the animal’s death, and parts were fixed in 10% buffered formalin. The lobe with trauma infliction and smaller control samples of the three other lobes were preserved. The lobe with inflicted injury was cut into thin slices (3 mm) for a thorough macroscopic and microscopic examination. For histologic examination, the samples were embedded in paraffin and stained using a hematoxylin and eosin and a standard Elastica-van Gieson protocol. An experienced pathologist assessed the degree of liver injury using light microscopy and photographs were taken (NanoZoomer; Hamamatsu Photonics, Herrsching, Germany).

2.7.

Statistical analysis

Statistical analyses were performed, and all graphs were created using GraphPad Prism version 6.0b for Mac (GraphPad Software, La Jolla, CA). All data passed the D’Agostino and Pearson omnibus normality test and are expressed as the means with standard deviation unless otherwise stated. A two-way analysis of variance model followed by a Sidak’s multiple comparisons test was used to analyze differences among groups. Differences over time (baseline versus hemodilution) within each group were analyzed by a repeatedmeasures model using analysis of variance with Tukey post hoc adjustment including groups and time as factors. Survival data were analyzed by the log-rank test. Statistical tests were performed two tailed and the level of significance was defined as P < 0.05.

3. 2.5.

Pathologic examination

Results

Blood sampling and analytical methods

Blood samples were taken at the following time points: “baseline” (after splenectomy), “hemodilution” (after hemodilution), “30,” “60,” and “120” (min after trauma); 120 min was also defined as the end of the experiment. Although the baseline measurements were obtained at 37 C, all the samples obtained at later time points (beginning with hemodilution) were examined at 33 C. A ball coagulometer with four channels (MC 4 plus; Merlin Medical, Lemgo, Germany) and suitable reagents from Dade Behring (Marburg, Germany) were used to measure fibrinogen concentration, prothrombin time (PT), and activated partial thromboplastin time (aPTT). A standard hematology analyzer

3.1. Laboratory parameters at baseline and after hemodilution/induction of hypothermia Baseline parameters, including blood cell count and coagulation measurements, were comparable between both groups with no significant differences (Tables 1 and 2, Fig. 1). Hemodilution and the contemporaneous induction of severe hypothermia (33.5 C  0.5 C; Fig. 2) significantly altered all coagulation parameters and led to a decrease in blood cell count. Fibrinogen levels (pooled from both groups) significantly decreased from 283 (76) to 112 (28) mg/dL (P < 0.001). PT was prolonged from 9.3 (0.6) to 14.7 (1.7) s, and the aPTT was prolonged from 11.2 (0.9) to 18.0 (2.1) s (P < 0.001 for all;

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Table 1 e Laboratory parameters.

Hemoglobin, g/L Control FT-patch Platelet count, 109/L Control FT-patch Fibrinogen, mg/dL Control FT-patch aPTT, s Control FT-patch

Baseline

Hemodilution

30 min after trauma

60 min after trauma

120 min after trauma

8.0  0.5 7.7  0.4

6.9  0.5* 6.6  0.2*

4.2  0.6y 5.0  0.6

3.6  0.7y 4.8  0.6

3.2  0.8y 4.9  0.5

285  62 252  45

126  20* 121  19*

92  28 104  19

87  22 104  14

73  27 105  17

268  62 297  89

105  20* 120  33*

63  16 89  31

62  16 90  32

56  13 100  31

11.2  1.0 11.2  0.9

18.0  1.6* 18.0  2.6*

21.8  3.4 19.5  3.2

21.9  2.7y 18.6  2.6

23.9  4.9y 16.8  1.2

Data are shown as the mean  standard deviation. * P < 0.001 within group (baseline versus hemodilution). y P < 0.05 FT-patch versus control.

Fig. 3, Table 1). Concordantly, thromboelastometric values showed a significant prolongation of CT and CFT, whereas MCF decreased significantly over time (P < 0.001 for all; Fig. 1).

3.2.

Laboratory parameters after trauma

After trauma and volume resuscitation, the blood cell count dropped in all animals. Hemoglobin concentrations, platelet counts, and fibrinogen concentrations continuously decreased in the control group until the end point, whereas these values remained stable in the FT-patch group (Table 1). Fibrinogen concentrations in the control group reached their lowest concentrations 120 min after trauma (56  13 mg/dL). Both the PT and aPTT showed a higher prolongation after trauma in control animals compared with the FT-patch group and continued to lengthen in the control group over time. In

opposition to these findings, FT-patchetreated animals showed a reduction both in the PT (17.3  3.9 s) and aPTT (19.5  3.2 s) between 30 and 120 min after trauma (PT: 16.1  2.9 s; aPTT: 16.8  1.2 s) with significant differences between groups (Fig. 3, Table 1). Both CT and CFT were prolonged in the control group in contrast to the FT-patch group. MCF was slightly reduced in the control group and stable in the FT-patch group (Fig. 1).

3.3.

Blood loss, hemodynamics, and survival

The amount of shed blood within 10 min after trauma before patch intervention (FT-patch versus control) was comparable between both groups (Fig. 4). The total blood loss at the end of the experimental phase differed significantly between the FTpatch (1195 [range 949e1407] mL; 36 [range 27e45] mL/kg) and

Table 2 e Hemodynamic variables.

Heart rate, beats/min Control FT-patch MAP, mmHg Control FT-patch CVP, mmHg Control FT-patch MPAP, mmHg Control FT-patch CO, L/min Control FT-patch

Baseline

Hemodilution

30 min after trauma

60 min after trauma

120 min after trauma

83  8 80  12

76  7 76  12

103  26* 78  18

115  29* 69  12

121  23* 62  9

89  10 81  11

80  10 79  8

39  7 51  9

38  10* 60  10

36  16* 65  10

72 63

81 82

52 62

52 62

52 62

18  2 17  3

21  3 21  3

13  3 17  5

14  4 17  4

16  4 19  5

4.6  0.7 4.5  0.4

4.5  0.8 5.0  1.0

2.6  0.8* 3.7  0.6

2.7  1.0* 4.0  0.8

2.5  1.2* 3.8  0.9

CVP ¼ central venous pressure; MPAP ¼ mean pulmonary artery pressure. Data are shown as the mean  standard deviation. * P < 0.05 FT-patch versus control.

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A 150

FT-patch

Control

P < .001

P = .002

P < .001

Clotting time (s)

125

100

75

50

25

Baseline

B

Hemodilution

10 min

30 min

60 min

120 min

500

Control

FT-patch

Clot formation time (s)

P = .001

400 P < .001

300

200

100

0

Baseline

Maximum clot firmness (mm)

C

Hemodilution

80

10 min

30 min

60 min

120 min

FT-patch

Control

70

60

P < .001

P < .001

50

40

30

Baseline

Hemodilution

10 min

30 min

60 min

120 min

Fig. 1 e Parameters of thromboelastometry: The CT (A), CFT (B) and the MCF (C) are shown at various time points (i.e., minutes after trauma). The results are presented as box plots (minimum, first quartile, median, third quartile, maximum).

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37

P < .001

Control

4000

FT-patch

P < .001

3000

Blood loss (mL)

Temperature (°C)

36

35

34

33

2000

P = .388

BL

HD

trauma

1000

30

60

120

minutes after trauma

0 Control

Fig. 2 e Blood temperature constantly measured using a pulmonary artery catheter at baseline (BL: after splenectomy), after hemodilution (HD) with contemporary induction of severe hypothermia, and at various time points after trauma. Blood temperature significantly dropped during hemodilution in both groups and was maintained between 33 C and 34 C. Data presented as the mean ± standard deviation.

the control group (2495 [range 1993e3344] mL; 74 [range 61e96] mL/kg; both P < 0.001). All measured hemodynamic parameters were comparable between the groups at baseline and after hemodilution (Table 2). After liver injury, the heart rate of control animals increased and almost reached a 2-fold value compared with the FT-patch group at the end of the study. Correspondingly, the MAP continued to fall in control animals over time, whereas FT-patchetreated animals showed an increase over time. CO declined significantly due to trauma in both groups and remained stable at the diminished levels over time with

35

Control

FT-patch

Prothrombin time (s)

30

*

25

20

FT-patch

10 min trauma

Control

FT-patch

total blood loss

Fig. 4 e Blood loss 10 min (before treatment) and 120 min after trauma. Horizontal lines represent the means ± standard deviations. Treatment with TachoSil (FT-patch) significantly reduced blood loss compared with control.

significant differences between the groups. Central venous pressure and mean pulmonary artery pressure remained stable at reduced levels after trauma. All animals in the FT-patch group survived the observational phase, whereas two of nine animals (P ¼ 0.145) in the control group died 90 and 104 min after the infliction of trauma.

3.4.

Macroscopic and microscopic analysis

Postmortem gross sectioning of the inflicted liver lobe showed comparable depth of lesions with 70%e90% of lobe thickness and laceration of veins of 3e4 mm in diameter in both groups. Microscopic evaluation confirmed parenchymal injury and comparable laceration of venous vessels. The FT-patch showed a tight adherence to the liver surface macro- and microscopically at the complete circumferential margins of the damaged liver and, therefore, covered the lesion completely (Fig. 5).

* P < .001

4.

15

BL

HD

trauma

10 30

60

120

minutes after trauma

Fig. 3 e PT measured on a coagulometer at baseline (BL), after hemodilution (HD), and at various time points after trauma. Data presented as the mean ± standard deviation. *P < 0.05 FT-patch versus control. PT significantly prolonged after HD in both groups and continued to lengthen in control group.

Discussion

This experimental animal trial is the first to examine the effects of TachoSil on blood loss and coagulation parameters after blunt liver injury under severe hypothermia in a coagulopathic pig model. TachoSil was effective in reducing blood loss and attenuating coagulopathy that resulted from the combination of hypothermia, hemodilution, and trauma. The combination of injury, coagulopathy, and hypothermia induced in the present study mimics the clinical reality of the hypothermic, coagulopathic, and bleeding patient who is in dire need of quick hemorrhage control [9,31]. A leading tenet of damage control surgery is fast control of hemorrhage to prevent death from exsanguination. Rapid bleeding control

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Fig. 5 e Representative section of liver tissue after an injury with a force of 236 N. The injury reaches deep into the parenchyma by approximately 70%e90% without penetration of the liver lobe (see parenthesis, picture A, left side). In general, a damage of veins of up to 3e4 mm could be found (see asterisk, picture A, left side). The TachoSil patch totally covers the area inflicted (see arrows, picture A, left side) and seals the margins tightly (see arrows, picture B, right side). Macroscopical photograph on the left side (A); microscopical photograph on the right side (B) (35, hematoxylin and eosin stain). (Color version of figure is available online.)

reduces the need for volume resuscitation, including transfusion of blood products, and aids to avert further deterioration of coagulopathy [32]. However, diffuse bleeding from traumatized areas of organs continues to be a challenging task in surgery [33]. Incompressible sources of hemorrhage and the potentially fatal combination of coagulopathy and hypothermia after severe trauma make this task even more difficult to accomplish [34]. Therefore, all available resources, as well as standard and additive techniques, such as local hemostatics like TachoSil, may be considered by emergency teams in these critical and time-sensitive cases [35]. TachoSil is broadly used in civilian surgical care and many advantageous applications of TachoSil in different surgical interventions have been reported [16,17]. The efficacy of TachoSil in the field of hepatic surgery was presented first by a prospective multicenter randomized clinical trial by Frilling et al. [36] and showed the significant superiority of TachoSil over argon beam coagulation after hepatic resection with regard to time until hemostasis was established. A more recent and identical randomized clinical trial confirmed these findings [37]. Another clinical trial demonstrated significant improvements of postoperative parameters such as drainage volume, transfusion requirement, rate of complications, and length of hospital stay with TachoSil during major and minor hepatectomies [38]. As all three clinical studies included patients for elective hepatic surgery only, no conclusions about the effectiveness of TachoSil under coagulopathy and/or hypothermia can be drawn. In addition to TachoSil, different commercially available and custom-made local hemostatic dressings and sealants feasible for application in trauma settings have been investigated in the past [35]. These studies were performed in large part by U.S. Military research groups and focused on commonly used hemostatic agents applicable in combat injuries. Arnaud et al. [39] compared 10 hemostatic dressings to a standard compressed gauze dressing in a noncoagulopathic porcine transection model of the femoral vasculature to simulate battlefield wounds with multiple vessel injuries under normothermia. The authors found only four dressings to be more effective in achieving bleeding control and improving survival rates compared with a standard gauze bandage. They identified absorbent property, flexibility, and

the hemostatic agent itself as the key factors for the efficacy. Another trial evaluated four preselected hemostatic dressings presumably more suitable for battlefield application to arrest bleeding in a porcine model of arterial injury in the groin [40] without preceding coagulopathy under normothermic conditions. The investigators found Combat Gauze to be most effective in reducing the amount of hemorrhage and allowing the highest rate of survival. This gauze is impregnated with a contact pathway activating clotting agent (kaolin). As the authors discussed, the dressing’s efficacy is dependent on normal coagulation function and may, therefore, be less effective in patients with a deteriorated coagulation status. On the other hand, a recently published experimental study observed a trend toward reduced blood loss, lower resuscitation requirements, and less mortality after the treatment with Combat Gauze in a highly lethal swine liver injury model under hypothermia and moderate coagulopathy compared with standard abdominal packing [41]. Hemostatic patches with a dried layer of human fibrinogen and human thrombin are independent of the coagulation status. Such patches have also been investigated for efficacy to control coagulopathic bleeding and prevent death in a model of grade V liver injury under hypothermia and were superior to standard hepatic packing [42]. The dry fibrin sealant dressing (DFSD) used by Holcomb et al. [43] was also coated with human fibrinogen and human thrombin dried onto a vicryl mesh backing. The DFSD enabled rapid hemorrhage control and improved survival in swine with severe liver injury complicated by dilutional and hypothermic coagulopathy compared with conventional liver packing or immunoglobulin G placebo sealant dressings. Notably, the concentrations of coagulation factors in DFSD were much higher compared with those of the TachoSil patches used in our study (DFSD versus TachoSil [15]: 15 versus 5.5 mg/cm2 human fibrinogen; 37.5 versus 2.0 IU/cm2 human thrombin). Despite these considerably lower concentrations, TachoSil was still highly effective in reducing blood loss in the present study under severe hypothermia. Hypothermia is known to alter coagulation [19e21] and is common in severely injured patients [44,45]. Hypothermia is one component of the “lethal triad” in traumatized patients along with acidosis and coagulopathy [45e47] and contributes to poor outcomes [47,48]. Martini et al. [49] identified acidosis

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and hypothermia as independent contributors to coagulopathy in swine. Although hypothermia delayed the onset of thrombin generation, acidosis primarily lowered the rates of thrombin generation rates. The “triad of death” is difficult to control or reverse because the different constituents of this condition interrelate and potentiate each other [50]. The degree of hypothermia and the likelihood for hemorrhage are in a dose-response type of relationship [20,51]. The coagulopathy provoked in the presented model is also multicausal. Dilutional and hypothermic effects impairing the activity of enzymes in the clotting cascade add up and alter the animal’s coagulation system. Despite the combination of severe hypothermia and dilutional coagulopathy in our present study, the application of the FT-patch mediated the rapid control of hemorrhage and, therefore, significantly reduced blood loss and improved survival. These main findings are similar to the data from two other studies investigating patches coated with higher concentrations of fibrinogen and thrombin with [43] and without [52] hypothermia, as well as our previously published study without hypothermia [18]. Next to the basic parameter of blood loss as a primary marker for the efficacy of TachoSil, we observed concordant changes in hemodynamic parameters despite severe hypothermia in our present study. The heart rates of FT-patchetreated animals were significantly lower, whereas the MAP and CO were significantly higher over the course of time, demonstrating a more severe and sustained hemorrhagic shock in untreated animals. Similar findings have been described for less effective sealants in a study comparing different local hemostatics [40]. In the present study, control animals with ongoing bleeding had a decrease in cell counts, fibrinogen concentrations, and prolongation of the PT and aPTT as global coagulation parameters. These findings are similar to our previous results as well with those described by Holcomb et al. [43]. In addition to these standard clotting assays, we included thromboelastometry in the coagulation analyses. Thromboelastometry is a rapidly evolving methodology used in the trauma setting [53] and gives a broader picture of the functionality of blood clot building. Thromboelastometric measurements revealed significant differences between the two groups in our study with prolonged CTs, prolonged CFTs, and decreased MCF in control animals compared with stable parameters in the FT-patch group. These findings indicate a continuous aggravation of coagulopathy if bleeding cannot be controlled and are similar to our data previously published, despite the induction of severe hypothermia. Some limitations to our study should be noted. The animals were hemodiluted before infliction of trauma to standardize the degree of resulting coagulopathy. Clinically, iatrogenic hemodilution results from blood loss followed by the replacement of shed blood with factor and platelet poor resuscitation fluids such as crystalloids and colloids. In addition, liver injury was induced in anaesthetized healthy pigs, preventing physiological responses to pain and inflammation that may have additional effects on hemostasis. In conclusion, this study shows TachoSil to be effective in rapid hemorrhage control in hypothermic and coagulopathic pigs and suggests it as a supportive alternative treatment in terms of damage control surgery and resuscitation.

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Acknowledgments The authors thank Thadda¨us Stopinski (Institute of Laboratory Animal Science), Renate Nadenau, and Christian Beckers (both Department of Anesthesiology) for their excellent support. Conflict of interest disclosures and funding: R.R. has received honoraria for lectures and consultancy from CSL Behring (Germany) and Novo Nordisk (Germany). R.T. is a consultant for Nycomed (Germany) and has given invited lectures. O.G. has received research funding from CSL Behring (Germany), Novo Nordisk (Denmark), Biotest (Germany), Nycomed (Denmark), and consultancy fees from Boehringer Ingelheim (Germany) and Bayer Healthcare (Germany). This research project was supported by an unrestricted educational grant from Nycomed (Roskilde, Denmark). The sponsor had no influence on the design of the study or the interpretation of results. The authors declare that they have no other competing interests.

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