Koller et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2021) 29:57 https://doi.org/10.1186/s13049-021-00867-5 ORIGINAL RESEARCH Open Access Normalization of blood clotting characteristics using prothrombin complex concentrate, fibrinogen and FXIII in an albumin based fluid: experimental studies in thromboelastometry Tobias Koller1* , Nadia Kinast2, Andres Guilarte Castellanos3, Sergio Perez Garcia1, Pilar Paniagua Iglesias1, Xavi León Vintro1, Jose Mateo Arranz1, Noelia Vilalta Seto1, Ma. Victòria Moral García1, Ana Belén Moreno-Castaño4, Jose Aznar-Salatti5, Gines Escolar Albaladejo4 and Maribel Diaz-Ricart4 Abstract Background: Colloid fluids supplemented with adequate combinations of coagulation factor concentrates with the capability to restore coagulation could be a desirable future treatment component in massive transfusion. Methods: Starting from a coagulation factor and blood cell-free albumin solution we added Prothrombin Complex Concentrate, Fibrinogen Concentrate and Factor XIII in different combinations and concentrations to analyze their properties to restore thromboelastometry parameters without the use of plasma. Further analysis under the presence of platelets was performed for comparability to whole blood conditions. Results: Albumin solutions enriched with Fibrinogen Concentrate, Factor XIII and Prothrombin Complex Concentrate at optimized concentrations show restoring coagulation potential. Prothrombin Complex Concentrate showed sufficient thrombin formation for inducing fibrinogen polymerization. The combination of Prothrombin Complex Concentrate and Fibrinogen Concentrate led to the formation of a stable in vitro fibrin clot. Fibrinogen and Factor XIII showed excellent capacity to improve fibrin clot firmness expressed as Amplitude at 10 min and Maximal Clot Firmness. Fibrinogen alone, or in combination with Factor XIII, was able to restore normal Amplitude at 10 min and Maximal Clot Firmness values. In the presence of platelets, the thromboelastometry surrogate parameter for thrombin generation (Clotting Time) improves and normalizes when compared to whole blood. Conclusions: Combinations of coagulation factor concentrates suspended in albumin solutions can restore thromboelastometry parameters in the absence of plasma. This kind of artificial colloid fluids with coagulation- restoring characteristics might offer new treatment alternatives for massive transfusion. (Continued on next page) * Correspondence: tkoller@santpau.cat 1 Hospital de la Santa Creu i San Pau, Universidad Autonoma de Barcelona, Departamento de Cirugía, Carrer de Sant Quintí, 89, 08041 Barcelona, Spain Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Koller et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2021) 29:57 Page 2 of 11 (Continued from previous page) Trial registration: Study registered at the institutional ethic committee “Institut de Recerca, Hospital Santa Creu i Sant Pau, with protocol number IIBSP-CFC-2013-165. Keywords: Massive bleeding, Blood transfusion, Fibrinogen, Factor XIII, Prothrombin complex concentrate, Fluid therapy, Hemostatic resuscitation, Plasma substitutes Background maintenance of the stabilizing effects on coagulation and Massive bleeding can cause severe intravascular hypovol- hemodynamics. emia with significant hypoperfusion of peripheral tissues We hypothesized, that an optimized and well-balanced leading to life-threatening hemorrhagic shock. The cor- combination of different coagulation factors, reconstituted rection of the underlying hypovolemic state with con- in an albumin-based carrier solution, would provide basic ventional resuscitation fluids like crystalloid or colloid clotting characteristics with TEM responses compatible solutions contributes to bleeding associated coagulopa- with whole blood if tested under the presence of platelets. thies by dilution of plasma coagulation factors [1]. De- In this study, we used a thromboelastometric approach terioration of physical clot strength and, in a later stage to analyze modifications of viscoelastic parameters in a of prolonged bleeding, reduced thrombin generation po- plasma-, and blood cell-free environment. It is technic- tential are the principal coagulopathic patterns found ally feasible to perform thromboelastometric analysis in when monitored by viscoelastic testing [2]. Viscoelastic such conditions, although this approach has been limited testing, like rotational thromboelastometry (TEM), is fre- to its use in laboratory investigations [12, 13]. quently used as a point-of-care tool in severe bleeding, providing comprehensive information about the visco- Methods elastic and temporal characteristics of blood clot forma- Experimental design tion [3]. Thereby identified coagulopathies caused by This study (protocol number IIBSP-CFC-2013-165) was de- single- or combined coagulation factor deficits are in- signed to explore in vitro the capability of CFCs to restore creasingly treated with coagulation factor concentrates coagulation properties. A series of experimental studies (CFC), like Fibrinogen Concentrate (FC), Prothrombin were performed to define the optimal factor concentrations Complex Concentrate (PCC) and Factor XIII Concen- of such coagulation resuscitating fluid (CRF). Starting from trate (FXIIIC) [4–6]. The principle goal of CFC-based a coagulation factor and blood cell-free solution of 5% hu- treatment strategies is to reduce transfusion rates of man albumin we added PCC, FC and FXIIIC in different allogeneic blood products and adverse events associated combinations and concentrations to analyze their proper- with plasma transfusion [7]. There is growing scientific ties to restore thromboelastometric parameters without the evidence that coagulation factor deficiencies in bleeding use of plasma. The optimal CFC composition was further patients can be effectively and safely treated with CFCs analyzed under the presence of platelets to improve com- with some studies showing better outcomes for CFCs parability to whole blood conditions. when compared to plasma transfusion [8–10]. By con- All coagulation factors – fibrinogen (FGN), factor XIII trast there is a certain paucity of high-quality evidence (FXIII) and prothrombin complex (PC) factors II, VII, IX in favor of plasma, although plasma transfusion is still a and X - were derived from commercial CFCs (FC, FXIIIC widely accepted standard of trauma and non-trauma and PCC). The optimal concentration of FGN and FXIII massive transfusion protocols [11]. The high acceptance in CRF was determined by direct comparison to clot firm- of plasma transfusion among many physicians might be ness parameters of plasma from an internal control group. explained, not only by its stabilizing effect on the coagu- The optimal concentration of PC coagulation factors was lation system providing a close-to-physiological factor determined by the shortest obtained clotting time (CT) composition but also by its resuscitation fluid quality value. CRF was considered as having a coagulation restor- with good intravascular volume effects in patients with ing potential to allow consideration as plasma substitute, hemorrhagic shock. Currently, no alternative products if thromboelastometric responses of the final CRF com- are available which share these two characteristics with position lay within the normal range for whole blood human plasma. Therefore, colloid fluids providing when tested in presence of platelets [14]. adequate intravascular volume effects combined with maintained hemostatic properties could be an interesting Plasma reference values for thromboelastometry future treatment component in massive transfusion and parameters damage control resuscitation, helping to reduce plasma TEM parameters were determined from plasma from transfusion and its associated side effects under healthy volunteers to define the reference range for Koller et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2021) 29:57 Page 3 of 11 clotting time (CT), the amplitude at 10 min (A10) and Preparation of study samples maximum clot firmness (MCF) under blood cell-free Aliquots of the stock solution containing coagulation conditions. For this purpose, we collected blood sam- factors were added to the AFS within citrated tubes to ples from 33 healthy volunteers who had not taken reach the defined final factor concentration. The study medication affecting coagulation- or platelet-function samples were warmed to 37 °C in the provided warming in the last 10 days. A sample of 4.5 ml of blood was chamber of the ROTEM® machine before testing. drawn from each donor in citrated tubes (0.129 M) and centrifuged at 3200 rpm for 25 min. Plasma Variable CFC concentrations and platelet count supernatant was used for FIBTEM analysis. FIBTEM The effect of various combinations of CFC-derived co- was used to minimize viscoelastic signals associated agulation factors suspended in AFS on their functional with residual platelets after centrifugation. The 95% contribution to clot formation was evaluated by TEM. confidence interval of the obtained values was defined The study samples were distributed in different test as the reference range for plasma. The established series. Within the same test series the protein concentra- reference ranges were later used for comparison with tion of only one component (FGN, FXIII or PC), or TEM responses of CRF samples to determine the platelet count, was gradually modified while the concen- optimized CRF factor concentration. tration of the other components was left unchanged. The protein concentration of PC used in our in vitro-ex- periments is provided as IU/ml referring to the under- Study samples lying factor IX activity, being the PC reference protein. Composition of study samples Three main series of tests were performed combining Study samples were composed of an artificial fluid solu- different CFC-derived proteins added to the AFS: tion (AFS) based on 5% human albumin together with a) Increasing PC concentrations (0.05, 0.1, 0.25, 0.5, 1, different CFC combinations and concentrations. 20% hu- 2 and 4 IU/ml) over a fixed FGN concentration of 4 g/l; man Albumin (Grifols®, Spain) was diluted with an iso- b) Increasing FGN concentrations (0.5, 1, 2, 4, 8 and 12 tonic, balanced, crystalloid solution (Viaflo Plasmalyte® g/l) over a fixed PC concentration of 1 IU/ml; c) Increas- 148, Baxter, Spain) to a final albumin concentration of ing FXIII concentrations (0.1, 0.5, 1, 2, 4 and 8 IU/ml) 5% correspondent to high physiologic plasma concentra- over a fixed PC concentration of 1 IU/ml and a fixed tions for albumin. Calcium gluconate was added to FGN concentration of 4 g/l. Each concentration step of achieve a physiological free ionized calcium concentra- the changing factor component defined one study tion of 1.0–1.2 mmol/l. The solution was buffered with sample that was tested by TEM. The study samples were TRIS buffer (1 M) to a physiological pH range between analyzed for viscoelastic properties with the FIBTEM-S 7.36–7.45. Electrolyte concentrations and pH were mea- subtest. A TEM response within the defined reference sured on the blood gas analyzer Radiometer®ABL 90 Flex values (Table 1) for A10 and MCF derived from internal to confirm the physiological composition of our stem controls determined the optimal FGN and FXIII com- solution. position of the CRF. Combined FGN/FXIII preparations All factor concentrates were provided by CSL Behring. with TEM responses within the normal range for A10/ The different coagulation factor concentrates (CFC) MCF were given priority to single factor preparations were reconstituted in stock solutions. PCC (Beriplex, (FGN alone) for defining the final CRF composition. CSL Behring GmbH, Germany), FC (Riastap, CSL Beh- The shortest CT value obtained in a series with increas- ring GmbH, Germany) and FXIIIC (Fibrogammin/Clu- ing PC concentrations defined the final PC concentra- vot, CSL Behring GmbH, Germany) were used as CFCs tion of CRF. for this in vitro analysis. The lyophilized proteins were In a fourth test series the effect of platelets on samples reconstituted with the minimum amount of the accom- containing the final CRF composition of PC (1 IU/ml), panied provider’s solution necessary for protein dissolv- FGN (4 g/l) and FXIII (1 IU/ml) was evaluated adding an ing, resulting in final concentrations of 0.025 IU/μl for increasing number of washed platelets (12.5, 25, 50, 100, factor IX (FIX) as reference protein in PCC, 0.1 mg/μl 200 and 400 platelets 103/μl) obtained from healthy, for FGN, and 0.05 IU/μl for FXIII. High final protein non-medicated donors. The samples were analyzed with concentrations in the stock solutions were necessary to the FIBTEM-S and EXTEM-S subtests. avoid dilutional effects during the preparation of the final study samples. The stock solution was directly used Preparation of platelet suspensions or stored at − 70 °C for later use. The final composition Blood was collected into citrate/phosphate/dextrose of AFS free of proteins and blood cells was tested as a (final concentration of citrate of 19 mM) and centrifuged negative control with EXTEM and FIBTEM subtests as (120 x g for 15 min) to obtain platelet-rich plasma. described in the thromboelastometry section. Washed platelets were obtained by mixing PRP with Koller et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2021) 29:57 Page 4 of 11 Table 1 Reference ranges for standard TEM parameters CT, A10, MCF. Reference ranges are presented for plasma and whole blood. Plasma ranges are derived from an internal control group. Values from an external control group from previous studies are also highlighted for comparison. Whole blood reference ranges are shown for comparison with TEM results of CRF under presence of platelets. The recommended treatment thresholds are presented to provide a clinical context of the obtained TEM results Reference ranges Plasma (Internal control)a Plasma (External control)b Whole bloodc Treatment thresholdd CT (sec) 47–54 53–71 42–74 > 80 A10 (mm) 17–24 n.a. 43–65 < 7 MCF (mm) 18–26 17–35 49–71 < 14 a Values derived from fresh plasma from 33 healthy, non-medicated volunteers. Reference range corresponds to 95% confidence interval b Values derived from fresh plasma as published by Schörgenhofer et al. [13] c Values derived from whole blood as published by Lang et al. [14] d Treatment thresholds for whole blood EXTEM-CT and whole blood FIBTEM-A10/MCF as published by Schöchl and Schlimp [15]. and Ranucci et al. [16] equal volumes of citrate/acid citric/dextrose (93 mM so- dispersion graph. In the case of linear correlation the dium citrate, 7 mM acid citric, and 140 mM dextrose), Pearson coefficient for linear correlation was applied. pH 6.5 containing 5 mM adenosine and 3 mM theophyl- For interpreting our data p < 0.050.05 were considered line (CCD–AT) [17]. The final pellet was resuspended in significant. a Hanks’ balanced salt solution (136.8 mM NaCl, 5.3 mM KCl, 0.6 mM Na2HPO4, 0.4 mM KH2PO4, 0.2 mM Results NaH2PO4-2H2O) supplemented with dextrose (2.7 mM) Negative control of coagulation factor free AFS samples and NaHCO3 (4.1 mM), pH 7.2, and maintained for 50 No response was detected in FIBTEM or EXTEM sub- min at 37 °C before experiments were performed. Con- tests when performed on AFS that were not enriched centrated washed platelets were added to the study with coagulation factors. The tests resulted in an infinite samples to reach the established platelet counts. CT value and no clot formation could be detected. Thromboelastometry Normal range of plasma tested by TEM TEM analysis was performed on the ROTEM® delta ma- The 95% confidence interval of the TEM parameters de- chine (Rotation Thromboelastometry, TEM Inter- termined in 33 plasma samples of healthy volunteers national, Munich, Germany). Plastic cups were filled was defined as the “normal range” for plasma. The re- with 300 μl of pre-warmed (37 °C) solutions. FIBTEM-S/ sults are summarized in Table 1 as “Internal Control”. EXTEM-S subtests were used providing extrinsic coagu- The upper limit of normal for CT in our internal control lation activation with/without cytochalasin-based deacti- group was 53 s for EXTEM subtests and 54 s for FIB- vation of platelets. FIBTEM is essentially dependent on TEM subtests. The obtained plasma reference range for the FGN function. This test inhibits the platelet contri- A 10 and MCF was 17–24 mm and 18–26 mm, respect- bution to clot formation, leaving only the clotting pro- ively. Previously published reference ranges for standard teins. Thus, one can observe the contribution of TEM parameters for plasma and whole blood and usu- functional FGN to clot formation. A minimum of three ally accepted treatment thresholds are shortly summa- independent measurements on different, freshly pre- rized in Table 1 for comparison [13–16]. pared samples were performed for each concentration step. Thromboelastometry measurements were per- Combination of PC and FGN leads to fibrin clot formation formed immediately after combining the different pro- in a plasma and platelet free environment tein and/or cellular components of the study samples. The combination of PC and FGN in an artificial colloid Standard TEM parameters were obtained for statistical solution free from other blood components leads to the analysis: CT, A 10, and MCF. formation of a thromboelastometrically measurable fi- brin clot. The formed fibrin clots were stable as no sig- Statistical analysis nificant deterioration of its viscoelastic integrity was The 95% confidential interval defined the normal range observed during the 60 min TEM response. Figure 1 for the obtained TEM parameters of the internal control shows a typical TEM graph using a FGN concentration group: CT, A10, and MCF. Statistical analysis was per- of 4 g/l and PC at 1 IU/ml (using the concentration of formed on SAS® 9.3 Statistical Software. The correlation FIX as a reference). The viscoelastic properties of the analysis of the collected data was performed on basis of tested fluids at this coagulation factor combination (CT a dispersion graph for illustrating values of TEM param- 117 ± 20 s, A10 10.7 ± 0.6 mm, MCF 12 ± 1.7 mm) were eters in function of the corresponding dose. Linear cor- located outside the aspired, predefined normal values relation was analyzed on basis of the underlying (Table 1), especially CT was considerably prolonged. Koller et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2021) 29:57 Page 5 of 11 evident than those observed on CT. As shown in Table 2, a positive linear correlation between rising PC con- centrations and thromboelastometry clot strength pa- rameters was seen (Pearson correlation coefficient of rho = 0.65, 0.57 and p-values < 0.0013, 0.0063 for A10 and MCF respectively). According to these data, a concentration of 1 IU/ml PC for the final CRF composition was determined. Rising FGN concentrations improved clot strength related parameters at a fixed PC concentration of 1 IU/ml Based on the previous results, a fixed PC concentration of 1 IU/ml was chosen for this test series as it provided Fig. 1 Combination of PCC and FGN leads to fibrin clot formation in the shortest CT values in our model. As summarized in a plasma and platelet free environment. Figure is representative for Table 3, a negative non-linear correlation was seen be- fibrin clot formation under a combination of PCC at 1 IU/ml with FGN at 4 g/l. tween increasing FGN concentrations and measured CT values. This effect was more pronounced in the lower FGN range between 0.5 and 2.0 g/l and became less sig- Fibrin formation was dependent on the presence of PC nificant with higher FGN concentrations above 2 g/l. as negative controls without PC proteins ruled out spon- Moreover, FGN concentrations rising from 0.5 to 12 g/ taneous fibrin formation during TEM analysis. l improved the TEM parameters that characterize the clot strength with a strong positive correlation between Increasing concentrations of PC caused progressive FGN concentrations and the corresponding TEM pa- improvements in TEM parameters at a fixed FGN rameters (Pearson correlation coefficient of rho = 0.98 concentration of 4 g/l and p-values of < 0.0001 for both A10 and MCF respect- As described in Table 2, a progressive shortening of CT ively, Table 3). At a physiological FGN concentration of was observed with increasing concentrations of PC. An 4 g/l the measured TEM responses (11/12 mm for A 10/ inverse association between PC concentration and CT MCF) were slightly below the desired TEM range of 17 was observed within the 0.05–1 IU/ml concentration mm. The final FGN concentration for CRF composition range with the shortest CT detected at a concentration of 4 g/l was determined in synopsis with the results of 1 IU/ml and an average CT of 117 s. This value was achieved in combination with FXIII. significantly prolonged when compared to the CT refer- ence range of our internal control group, to external Increasing concentrations of FXIII (0.1, 0.5, 1, 2, 4, 8 IU/ml) controls, and published treatment thresholds (Table 1). enhanced clot strength at fixed concentrations of PC (1 Higher PC concentrations (> 1 IU/ml) did not further IU/ml) and FGN (4 g/l) shorten CT values. Even in very high concentrations like Fixed concentrations of FGN (4 g/l) and PC (1 IU/ml) 4 IU/ml, CT did not further improve. were used in this experimental setting. FXIII effects on Effects of increasing concentrations of PC on fibrin fibrin clot strength were evaluated by A 10 and MCF. As clot strength measured by A 10 and MCF were less shown in Table 4, a moderate to high positive Table 2 Analysis of viscoelastic parameters in albumin-based colloid solutions enriched with prothrombin complex concentrate (PCC) and fibrinogen (FGN). Different PCC concentrations (0.05, 0.1, 0.25, 0.5, 1, 2, 4 IU/ml) were combined with fixed fibrinogen (FGN) concentrations of 4 g/l. The albumin concentration was maintained stable at 5%. Negative controls on PCC-free solutions did not show any measurable ROTEM response (infinite CT). A minimum of three repeats for each concentration step was performed. The value in bold letters is prolonged (>80s) but still reflects the optimal PCC response for CRF composition Increasing PCC concentrations over FGN 4 g/l PCC conc.a 0,05 0,1 0,25 0,5 1 2 4 b,* CT 613 ± 251 337 ± 46 216 ± 50 168 ± 22 117 ± 20 95 ± 40 115 ± 18 A10c,** 5.7 ± 1.5 6.3 ± 1.5 6.7 ± 0.6 8.3 ± 1.5 10.7 ± 0.6 14 ± 4.3 11.7 ± 2.1 MCFc,*** 6.3 ± 1.5 7.3 ± 2.1 7±1 9.3 ± 2.3 12 ± 1.7 14.3 ± 4.9 12 ± 2.6 *p < 0,0001, Pearson rho 0,9 for1/CT, **p = 0,0013, Pearson rho 0,65, ***p = 0,0063, Pearson rho 0,57 a PCC concentrations in IU/ml refer to final factor IX concentrations as reference protein in this product b CT in seconds. Mean values ± SD c A10 and MCF in mm. Mean values ± SD Koller et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2021) 29:57 Page 6 of 11 Table 3 Analysis of viscoelastic parameters in albumin-based investigated under the previously established conditions colloid solutions enriched with fibrinogen (FGN) and using fixed CRF concentrations of PC (1 IU/ml), FXIII prothrombin complex concentrate (PCC). Different FGN (1 IU/ml) and FGN (4 g/l) as this combination provided concentrations (0.5, 1, 2, 4, 8, 12 g/l) were combined with fixed optimal values for CT and clot strength in TEM studies. PCC concentrations of 1 IU/ml. The albumin concentration was The presence of platelets (in the optimally designed maintained stable at 5%. Negative controls on fibrinogen free CRF) significantly improved fibrin clot strength parame- solutions did not show any measurable ROTEM response (infinite CT). A minimum of three repeats for each concentration ters assessed by A10 and MCF (Fig. 2a and b). A moder- step was performed. The values in bold letters reflect the ate to high positive correlation was observed between optimal FGN response for CRF composition rising platelet concentrations and A10 and MCF mea- Increasing FGN concentrations over PCC 1 IU/ml surements (Pearson correlation coefficient of rho = 0.89/ 0.86, respectively and p-values of 0.0001). Clot strength FGN 0,5 1 2 4 8 12 conc. a values reached the normal range for EXTEM whole CT b,* 4032 ± 620 ± 620 221 ± 90 117 ± 20 154 ± 27 198 ± 1 blood under a minimum platelet concentration around 1949 100 × 103/μl, as calculated from a regression analysis. No A10c,** n.a. 2.7 ± 0.6 3.6 ± 0.6 10.7 ± 0.6 25.7 ± 4.2 47 ± 1.4 effect on clot strength was observed for platelets in the FIBTEM tests performed in parallel. MCFc,*** n.a. 3.7 ± 0.6 3.3 ± 0.6 12 ± 1.7 26.3 ± 3.1 49.5 ± 3.5 As shown in Fig. 2c, the presence of platelets (in the *p < 0,0001, Pearson rho 0,84 for1/FGN, **p < 0,0001, Pearson rho 0,98, ***p < 0,0001, Pearson rho 0,98 optimal CRF) shortened CT. A moderate to high nega- a b FGN concentrations in g/l tive correlation was observed with rising platelet concen- CT in seconds. Mean values ± SD c A10 and MCF in mm. Mean values ± SD trations and measured CT (Pearson correlation coefficient of rho = − 0.80, p < 0.0001). TEM values correlation was observed for the Pearson correlation coef- reached normal whole blood CT values for EXTEM sub- ficient between rising FXIII concentrations and A10 and tests under a minimum platelet concentration of around MCF (p-values of 0.004 and 0.002, respectively). The pre- 100 × 103/μl. viously observed TEM response at a FGN concentration As shown in Table 5, CRF at PC 1 IU/ml, FGN 4 g/l, of 4 g/l (MCF 12 mm) in a FXIII-free environment (MCF FXIII 1 IU/ml reached TEM parameters comparable to 12 mm), significantly improved to MCF of 24 mm by add- whole blood EXTEM reference ranges when tested ing 1 IU/ml of FXIII, thus reaching the upper limit of our under the presence of 100 × 103/μl platelets. predefined range for normality. No additional effect or statistical correlations were observed for other tested Discussion TEM parameters (CT). According to the combined data Data from our in vitro study demonstrate that it is pos- on FGN and FXIII, final concentrations of 4 g/l for FGN sible to restore coagulation properties by combining de- and 1 IU/ml for FXIII were determined for further analysis fined concentrations of coagulation factor concentrates of CRF under the presence of platelets. in an albumin-based colloid solution. The viscoelastic clot formation parameters A 10 and MCF observed in Platelets completely restore whole blood TEM CRF were comparable not only to human plasma, but parameters, including CT also to whole blood under the presence of platelets. The The impact of increasing platelet counts (12.5, 25, 50, surrogate parameter for thrombin generation, CT, was 100, 200, 400 × 103/μl) on various TEM parameters was prolonged when compared to our internal control group, Table 4 Analysis of viscoelastic parameters in albumin-based colloid solutions enriched with factor XIII (FXIII), prothrombin complex concentrate (PCC) and fibrinogen (FGN). Different FXIII concentrations (0.1, 0.5, 1, 2, 4, 8 IU/ml) were combined with fixed PCC and FGN concentrations of 1 IU/ml and 4 g/l, respectively. The albumin concentration was maintained stable at 5%. Negative controls for FXIII- free solutions are represented by the corresponding compositions shown in Tables 2 and 3. A minimum of three repeats for each concentration step was performed. The values in bold letters reflect the optimal FXIII response for CRF composition Increasing FXIII concentrations over PCC 1 IU/ml and FGN 4 g/l FXIII conc. a 0.1 0.5 1 2 4 8 b,* CT 123 ± 12.7 128 ± 17.3 135.3 ± 12.7 122.3 ± 7.5 133.5 ± 29 133.3 ± 22.2 A10c,** 12.5 ± 0.7 16.3 ± 1.5 21.3 ± 2.1 24.3 ± 2.3 30.3 ± 11.4 30.7 ± 9.9 c,*** MCF 13.5 ± 0.7 18.7 ± 2.1 23.7 ± 3.1 26.7 ± 4.6 32.3 ± 10.4 35.7 ± 12.4 *no correlation, **p = 0,004, Pearson rho 0,65, ***p = 0,002, Pearson rho 0,69 a FXIII concentrations in IU/ml b CT in seconds. Mean values ± SD c A10 and MCF in mm. Mean values ± SD Koller et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2021) 29:57 Page 7 of 11 Fig. 2 Analysis of viscoelastic parameters of CRF, defined as albumin-based colloid solution containing PCC [1 IU/ml], FXIII [1 IU/ml] and FGN [4 g/l]). Different platelet concentrations (12.5, 25, 50, 100, 200, 400 × 103/μl) were combined with CRF. A minimum of three repeats for each concentration step was performed. The continuous line in the graph shows the lower limit of normal range for whole blood TEM parameters. a Effect of different platelet concentrations on A10. b Effect of different platelet concentrations on MCF. c Effect of different platelet concentrations on CT but reached normal levels for whole blood when ana- be an appealing new treatment component. Additional lyzed in presence of platelets at 100 × 103/μl [14]. Fluid point-of-care monitoring would still allow for goal- substitutes like CRF could be an interesting treatment directed top-up corrections, but monitoring intensity option, with clinical indications comparable to those of could be reduced. Easy storage of the CRF components fresh frozen plasma. at 4 °C, immediate availability without thawing time, and Massive bleeding, independent of its etiology (trauma, the universal applicability, independent of blood group obstetrical or surgical), usually includes high ratio FFP compatibility, could provide both logistic and clinical ad- transfusion guided by institutional massive transfusion vantages of this new product compared to frozen protocols, based on a large body of evidence favoring plasma. The clinical indications for CRF administration plasma against coagulation factor-free resuscitation could largely be analog to those of plasma, focusing on fluids [11, 18]. This hemostatic resuscitation concept, uncontrolled bleeding events related to multifactor defi- however, is insufficient to avoid massive transfusion- ciencies in hypovolemic patients, in which ongoing associated multifactor coagulopathies [19]. Factor con- factor-free fluid therapy could cause further deterior- taining fluids for volume therapy, like CRF, that are ation of the basic coagulation mechanisms. In these ur- characterized by optimized viscoelastic properties could gent scenarios CRF could provide shorter decision-to- treatment times than those known for plasma transfu- sion. CRFs could easily be held available, independent of Table 5 Comparison of whole blood reference ranges with TEM results for CRF under presence of 100 × 103/μl platelets blood bank facilities, in all areas exposed to massive bleeding scenarios, like operating theatres, ICUs, delivery WHOLE BLOODa CRF + PLATELETS rooms, emergency departments and even in prehospital CT (s) 42–74 74 +/− 5 emergency- or military settings. A 10 (mm) 43–65 45 +/− 3 Future clinical studies in massive transfusion scenarios MCF (mm) 49–71 59 +/− 2 will have to show if substituting plasma by CRF is feas- a Whole blood reference ranges as published by Lang et al. [14] ible and if equal efficacy in terms of hemodynamic and Koller et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2021) 29:57 Page 8 of 11 coagulation stability will be provided. Furthermore clin- albumin-based colloids seem to provide protective ef- ical trials will have to show if plasma-specific adverse fects against glycocalyx shedding. This albumin- events like “transfusion-related acute lung injury (TRAL mediated protection of the glycocalyx layer is currently I)” or “transfusion-related immune modulation (TRIM)” demonstrated in mostly preclinical, in-vitro studies, and would be less frequent under treatment of purified it remains a matter to future studies if this translates plasma-derived components like CFCs or albumin. It is into a clinically detectable advantage of albumin contain- not finally established from previous clinical studies, if ing resuscitation fluids [25, 26] . the overall complication rate, especially when compared Hemostasis is a result of coordinated interactions be- to solvent and detergent treated pooled plasma (S/D tween platelets and coagulation mechanisms [27]. Co- plasma), really results in a better safety profile for factor agulation mechanisms necessary for consolidation of concentrates. Next to these still to be answered clinical platelet mediated primary hemostasis require a cascade issues, current prices for the final CRF components of enzymatic reactions leading to the formation of fibrin would imply a major economic obstacle for the imple- [15, 28, 29]. Results of the present study indicate that co- mentation of this product into clinical routine, even if agulation mechanisms can be reproduced using a re- outcome superiority compared to plasma-based standard stricted number of coagulation factors suspended in a of care could be demonstrated in future clinical studies. neutral fluid. To our knowledge, this is the first experi- Human albumin 5% was chosen as carrier solution for mental study that has been able to demonstrate that the the coagulation factor compound of our CRF for differ- combination of commercially available CFCs in an initial ent reasons. First, colloids were favored against crystal- coagulation factor- and blood-cell-free solution leads to loids, because accurately defined coagulation factor the formation of a stable in vitro fibrin clot. The initi- concentrations within a predefined volume of a resusci- ation of the coagulation in this fluid requires the use of tation fluid would only make sense, if the underlying EXTEM or FIBTEM reagents whose components (cal- carrier showed adequate and sustained volume effects in cium, phospholipids and tissue factor) would trigger the the intravascular space. Second, the colloid should not activation of the prothrombin complex coagulation fac- interfere in a significant manner with the coagulation tors VII, IX, X and II contained in commercial PCCs [6]. system. Although all colloid solutions show dilutional ef- These coagulation factors lead to sufficient thrombin fects, albumin solutions, together with gelatins, seem to generation and warrant the basic activating mechanism cause less colloid-specific, detrimental effects on platelet of the coagulation system to sustain in vitro fibrin function or fibrin polymerization than other colloids, polymerization. FGN provides the structural clotting like dextrans, or starches [20, 21]. We preferred albumin substrate supporting secondary hemostasis [30]. The ne- against gelatins to optimize comparability to FFP in fu- cessary FGN concentration in CRF to reach normal ture trials. Experimental studies show that albumin- TEM values (when combined with FXIII) was found in based colloid solutions provide stabilizing effects on the the range of physiological plasma concentrations, around endothelial barrier and show intravascular plasma ex- 4 g/l for FGN and 0,5–1 IU/ml for FXIII. FXIII cross- pander effects of nearly 100% [22, 23]. By contrast, no links fibrin, completing blood coagulation and protecting such effects on the endothelial barrier could be demon- the hemostatic plug from the fibrinolytic activity at the strated for CFCs [24]. The administration of well- clot formation site. In vitro studies demonstrated that balanced coagulation factors in carrier solutions with supplementation with FXIIIC increases clot firmness constant intravascular volume effects might be a safe assessed by TEM in perioperative patients with elevated way to treat bleeding associated coagulopathies, as “over- FGN and reduced FXIII levels [31]. However, in another shot” peak plasma concentrations caused by the infusion in vitro model of massive transfusion in trauma, com- of highly concentrated factor formulas (as under non- bination therapies with FC and fresh frozen plasma, but diluted CFC administration) would be avoided. The not FXIIIC, improved both coagulation kinetics and intravascular volume effect of colloidal resuscitation fibrin-based clot strength [32, 33]. Our present study in- fluids seems to be context-sensitive and correlated to dicates that increasing concentrations of FXIII enhance the integrity of the endothelial glycocalyx layer. There is clot strength at fixed concentrations of PC (1 IU/ml) and growing evidence that in special clinical conditions like FGN (4 g/l). Consistently, there is further evidence that sepsis and trauma, characterized by elevated glycocalyx FXIII deficiency will impair FGN function and fibrin for- shedding rates, the volume expander rate of isooncotic mation, suggesting an inverse link between low FXIII colloids would be less than predicted. The underlying levels and enhanced thrombin generation, modifying the glycocalyx disruption seems to be partially driven by a structure-function relationship of fibrin to support “low-protein environment” caused by aggressive crystal- hemostasis [34]. Data derived from clinical studies loid or synthetical colloid fluid treatment. By contrast, propose maintenance of 50–60% of FXIII activity to protein containing resuscitation fluids like plasma or avoid bleeding tendency in the perioperative setting [35]. Koller et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2021) 29:57 Page 9 of 11 CRF compositions without FXIII, yielding comparable relevance. The currently scientific rationale rather sug- clot strength in TEM when compared to our final com- gests that PCCs might be associated to overshot throm- position, are possible from a theoretical point of view. bin generation with the potential to induce disseminated We decided to add a purified source of FXIII to our final intravascular coagulation and that Antithrombin III sup- CRF composition despite the high potential of plements might mitigate this potentially dangerous ad- concentrate-derived FGN on viscoelastic clot strength to verse effect [41]. The complete absence of antithrombin maintain a close-to-physiological factor composition. in the final CRF composition is a major limitation of our The safe upper limit of FC treatment has not been experiments and the effects of PCC supplements in clin- precisely defined. It is currently suggested that plasma ical situations with reduced antithrombin levels will have levels of FGN should reach 1.5 to 2 g/l in bleeding pa- to be analyzed in future trials. tients [36]. There is a clear tendency, as reported in dif- A further limitation of our experimental studies is the ferent guidelines, to recommend elevating plasma FGN complete absence of red blood cells. Red blood cells in some clinical situations [8, 37, 38]. Taking into con- seem to exert a more important role in primary sideration the results of our TEM studies it may be diffi- hemostasis, whereas their modulating effect on second- cult to maintain a well-balanced coagulation factor ary hemostasis seems to be negligible [13]. The fact is composition during a long-lasting, high-dynamic bleed- that, viscoelastic studies can be reliably performed in ing event if supplements are only point-of-care driven plasma samples [12, 13]. Surprisingly, an inverse relation and punctual. In this context, a fixed ratio of clotting between hematocrit and clot firmness was previously re- factors in CRFs administered under volume therapy ported under experimental and clinical anemic condi- could provide more balanced stability within the com- tions [42]. We cannot rule out that the presence of red plex multifactor system of blood coagulation than single blood cells in our in vitro model could lead to a measur- factor substitutes as proposed in current algorithms. able reduction of clot firmness parameters. However, fol- CT in TEM is partially dependent on thrombin gener- lowing reports of Schoergenhofer et al. [13] no effects ation. Direct anticoagulants reducing thrombin gener- on other TEM parameters should be expected under ation definitively prolong CT [39]. Platelets contribute to whole blood conditions. Under massive transfusion enhance thrombin generation, accelerate CT, and in- using CRF as a plasma substitute, transfusion of red crease MCF. Additionally, platelet phospholipids dra- blood cells would be an integral part of the clinical man- matically contribute to the amplification of coagulation agement to uphold an adequate amount of oxygen car- mechanisms, thus potentiating thrombin generation and riers within the circulating blood volume. fibrin polymerization. Fibrin then interacts with acti- Altogether, the transfer of our data into a clinical con- vated platelets and plays a critical role in MCF. CT text must therefore be done very carefully. All factor values of platelet-free CRF samples in our in-vitro exper- components of CRFs have previously been safely admin- iments were significantly prolonged when compared to istered in loose compositions for the management of plasma CT levels of our internal control group. Several bleeding associated coagulopathy [43]. PCCs show a reli- reasons may account for these findings: able safety profile and are now the treatment of choice First, our in vitro samples were completely free of any for the emergency reversal of Vitamin K antagonists [44, phospholipids or cell membrane fragments that could 45]. Nevertheless, a careful assessment of the thrombo- influence factor activation. Consequently, the addition of genic potential of fixed factor combinations for the treat- platelets to CRF containing 1 IU/ml PC, 4 g/l of FGN ment of a multiple factor deficit under massive bleeding and 1 IU/ml FXIII leads to the normalization of CT and will have to be performed in future studies. MCF. It could be assumed from our studies that, when combined with CRF, a platelet count around 100 × 103/ μl should be required to fully reconstitute TEM parame- Conclusions ters to levels observed in whole blood studies (see Fig. Coagulation factor concentrates suspended in albumin 2a-c). CT values above 80 s are considered to reflect solutions have the potential to restore mechanisms of pathological thrombin generation and are generally ac- secondary hemostasis in the absence of any blood com- cepted as treatment threshold. CT values of CRF com- ponent, showing viscoelastic properties comparable to bined with platelets were significantly shorter than this whole blood when tested in presence of platelets. Coagu- generally recommended treatment thresholds [15] lation factor enriched albumin-based colloids could be a (Tables 1 and 5). valuable tool to provide stable intravascular volume ef- Second, the used PCC in our experiments contains fects in hypovolemic conditions and to simultaneously heparin. Other study groups previously reported about maintain basic coagulation mechanisms. This could offer CT sensitivity of extrinsically activated TEM tests [40]. future alternatives to transfusion of fresh frozen plasma It is questionable if this phenomenon has any clinical under resuscitation conditions. Koller et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine (2021) 29:57 Page 10 of 11 Abbreviations 4. Korte WC, Szadkowski C, Gahler A, Gabi K, Kownacki E, Eder M, et al. Factor AFS: Artificial Fluid Solution; A10: Amplitude at 10 min; CFC: Coagulation XIII substitution in surgical cancer patients at high risk for intraoperative Factor Concentrate; CRF: Coagulation Resuscitating Fluid; CT: Clotting Time; bleeding. Anesthesiology. 2009;110(2):239–45. https://doi.org/10.1097/ALN. FC: Fibrinogen Concentrate; FGN: Fibrinogen; FXIII: Factor XIII; FXIIIC: Factor 0b013e318194b21e. XIII Concentrate; MCF: Maximum Clot Firmness; PC: Prothrombin Complex; 5. Colomina MJ, Lobo AD, Garutti I, Gomez-Luque A, Llau JV, Pita E. PCC: Prothrombin Complex Concentrate; TEM: Thromboelastometry Perioperative use of prothrombin complex concentrates. Minerva Anestesiol. 2012;78(3):358–68. Acknowledgements 6. Cappabianca G, Mariscalco G, Biancari F, Maselli D, Papesso F, Cottini M, Not applicable. et al. Safety and efficacy of prothrombin complex concentrate as first-line treatment in bleeding after cardiac surgery. Crit Care. 2016;20(1):5. Authors’ contributions 7. Pandey S, Vyas GN. Adverse effects of plasma transfusion. Transfusion. 2012; TK designed the study. PP, JM, JA and VM reviewed the study protocol. TK, 52(Suppl 1):65s–79s. NK, AG, SP, NV and AM collected and assessed the experimental data. TK, GE 8. Ranucci M, Baryshnikova E, Crapelli GB, Rahe-Meyer N, Menicanti L, Frigiola and MD interpreted the data and drafted the manuscript. PP, JM, XL, AM and A, Surgical Clinical Outcome REsearch (SCORE) Group. Randomized, double- JA reviewed the manuscript and provided critical comments for the blinded, placebo-controlled trial of fibrinogen concentrate supplementation interpretation and discussion of the data. XL, TK and GE performed the after complex cardiac surgery. J Am Heart Assoc. 2015;4(6):e002066. https:// statistical data interpretation. All authors read and approved the final doi.org/10.1161/JAHA.115.002066. manuscript. 9. Innerhofer P, Fries D, Mittermayr M, Innerhofer N, von Langen D, Hell T, et al. Reversal of trauma-induced coagulopathy using first-line coagulation Funding factor concentrates or fresh frozen plasma (RETIC): a single-centre, parallel- This work was partially supported by the Spanish government grants: DTS16/ group, open-label, randomised trial. Lancet Haematol. 2017;3026(17):1–14. 00133, PIE15/00027 (ISCIII) and FIS PI19/00888, and the Catalan government 10. van den Brink DP, Wirtz MR, Neto AS, Schochl H, Viersen V, Binnekade J, (grant number SGR 675-2017, AGAUR) and by CSL Behring, which has also et al. Effectiveness of prothrombin complex concentrate for the treatment provided reagents for the experimental work. of bleeding: a systematic review and meta-analysis. J Thromb Haemost. 2020;18(10):2457–67. https://doi.org/10.1111/jth.14991. Availability of data and materials 11. Malone DL, Hess JR, Fingerhut A. Massive transfusion practices around the The dataset used and/or analysed during the current study are available globe and a suggestion for a common massive transfusion protocol. J from the corresponding author on reasonable request. Trauma. 2006;60(6 Suppl):S91–6. https://doi.org/10.1097/01.ta.0000199549. 80731.e6. Declarations 12. Whiting D, Di Nardo JA. TEG and ROTEM: technology and clinical applications. Am J Hematol. 2014;89(2):228–32. https://doi.org/10.1002/a Ethics approval and consent to participate jh.23599. This study was approved by the Ethics Committee “Instituto de Recerca 13. Schoergenhofer C, Buchtele N, Schwameis M, Bartko J, Jilma B, Jilma- Hospital de la Santa Creu I Sant Pau” and registered under the protocol Stohlawetz P. The use of frozen plasma samples in thromboelastometry. number IIBSP-CFC-2013-165. Clin Exp Med. 2017;17(4):489–97. https://doi.org/10.1007/s10238-017-0454-5. Written informed consent was obtained from healthy volunteers before 14. Lang T, Bauters A, Braun SL, Potzsch B, von Pape KW, Kolde HJ, et al. Multi- blood sample extraction. centre investigation on reference ranges for ROTEM thromboelastometry. Blood Coagul Fibrinolysis. 2005;16(4):301–10. https://doi.org/10.1097/01.mbc. Consent for publication 0000169225.31173.19. Not applicable. 15. Schochl H, Schlimp CJ. Trauma bleeding management: the concept of goal- directed primary care. Anesth Analg. 2014;119(5):1064–73. https://doi.org/1 Competing interests 0.1213/ANE.0b013e318270a6f7. Tobias Koller received a study grant from CSL Behring. Pilar Paniagua 16. Ranucci M, Baryshnikova E. Fibrinogen supplementation after cardiac received lecture fees from CSL Behring. Jose Aznar-Salatti is working as CSL surgery: insights from the zero-plasma trial (ZEPLAST). Br J Anaesth. 2016; Behring Medical Affairs Manager at a Spanish Affiliate. The remaining authors 116(5):618–23. https://doi.org/10.1093/bja/aev539. have no conflict of interest to declare. 17. Lopez-Vilchez I, Galan AM, Hernandez MR, Caballo C, Roque M, Diaz-Ricart M, White JG, Escolar G. Platelet-associated tissue factor enhances platelet Author details reactivity and thrombin generation in experimental studies in vitro. Thromb 1 Res. 2012;130(6):e294–300. https://doi.org/10.1016/j.thromres.2012.10.003. Hospital de la Santa Creu i San Pau, Universidad Autonoma de Barcelona, Departamento de Cirugía, Carrer de Sant Quintí, 89, 08041 Barcelona, Spain. 18. Sommer N, Schnuriger B, Candinas D, Haltmeier T. Massive transfusion 2 protocols in nontrauma patients: a systematic review and meta-analysis. Consorci Sanitari Alt Penedés-Garraf, Carrer de l’Espirall, s/n, Vilafranca del Penedés, Spain. 3Hospital Mútua Terrassa, Plaça del Doctor Robert 5, 08221 J Trauma Acute Care Surg. 2019;86(3):493–504. https://doi.org/10.1097/TA. Terrassa, Barcelona, Spain. 4Hematopathology, Pathology Department, CDB, 0000000000002101. Hospital Clinic, IDIBAPS, University of Barcelona, Barcelona, Spain. 5CSL 19. Khan S, Brohi K, Chana M, Raza I, Stanworth S, Gaarder C, Davenport R. Behring S.A, Barcelona, Spain. Hemostatic resuscitation is neither hemostatic nor resuscitative in trauma hemorrhage. J Trauma Acute Care Surg. 2014;76(3):561–7; discussion 7-8. 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