Clauss Fibrinogen Research Articles

Viscoelastic haemostatic assays and fibrinogen concentration tests during haemodilution

BackgroundThe efficacy of concentrates of fibrinogen and factor XIII in hypothermia and haemodilution has not yet been completely investigated. Clauss fibrinogen measurement may overestimate fibrinogen concentrations during in vitro haemodilution with hydroxyethyl starch (HES).MethodsCoagulation was assessed with viscoelastic haemostatic assays [rotational thromboelastometry (ROTEM) or free oscillation rheometry (FOR)] in vitro. Fibrinogen measurement methods were compared in observational studies.ResultsHaemodilution with human albumin (HA) decreased the clot strength of ROTEM assay with tissue factor activation [EXTEM maximal clot formation (MCF)] less than the synthetic colloids HES or dextran (P < 0.001), but equal with crystalloid fluids [normal saline (NA) or Ringer's acetated solution (RA)]. Addition of fibrinogen increased the clot strength of ROTEM assay with tissue factor activation and platelet inhibitor cytochalasin D, i.e. fibrinogen‐dependent clot strength (FIBTEM MCF) more after haemodilution with HA than synthetic colloids (P < 0.001), but less than or equal with crystalloids. In haemodilution with HA, fibrinogen increased coagulation as measured with ROTEM dose dependently (P < 0.001). Factor XIII (FXIII) had no effect alone, but an additional effect to that of fibrinogen on clot strength, FIBTEM MCF (P < 0.02). Hypothermia at 33°C and haemodilution with HES, but not RA, interacted to decrease fibrinogen‐dependent clot strength measured with FOR (Fibscreen2 G′max, P < 0.001), and clot velocity measured with FOR (Fibscreen1 coagulation time 2, P = 0.035) or ROTEM (EXTEM clot formation time, P < 0.001). Fibrinogen (±FXIII) increased coagulation independently of temperature (33° or 37°C). After infusion of 1000 ml of HES to patients, fibrinogen measured with two Clauss methods and one immunological method decreased to the same extent (29%, 27% and 31%), whereas fibrinogen‐dependent clot strength (ROTEM FIBTEM MCF) decreased more (44%, P > 0.001). Finally, when comparing seven different Clauss methods during cardiac surgery, within‐method variability was low, but between‐method variability was high (mean difference > 0.5 g/l). No difference between pre‐ and post‐weaning from cardiopulmonary by‐pass was seen.ConclusionFibrinogen concentrate increased coagulation more after haemodilution with HA than synthetic colloids, but equal with or less than after crystalloid haemodilution. FXIII had an additional effect to that of fibrinogen. Hypothermia and haemodilution with HES interacted to decrease coagulation. Fibrinogen ± FXIII increased coagulation also at 33°C. Two Clauss methods after in vivo HES haemodilution did not overestimate fibrinogen; however, fibrinogen‐dependent clot strength decreased more than fibrinogen concentration. Between‐method variability with seven Clauss methods was high. These findings support the use of fibrinogen concentrate after resuscitation with HA, also at hypothermia, but question the use of colloids, especially HES, in resuscitation. Clauss fibrinogen methods need to improve.

Fibrinogen in trauma, an evaluation of thrombelastography and rotational thromboelastometry fibrinogen assays

BackgroundIdentifying hypofibrinogenemia in trauma is important. The optimal method of fibrinogen determination is unknown. We therefore evaluated fibrinogen levels determined by two whole blood viscoelastic hemostatic assays, thrombelastography functional fibrinogen (FF) and rotational thromboelastometry FIBTEM in trauma patients and compared these with the plasma-based Clauss method. Materials and methodsProspective study of consecutive adult trauma patients admitted to a level I trauma center. Levels of fibrinogen were analyzed by Clauss, FF, and FIBTEM on arrival. These methods were compared, and we then investigated whether specific cutoffs of fibrinogen levels were indicative for an increased risk of receiving a transfusion within the initial 6 h. ResultsA total of 182 patients with an Injury Severity Score of 17 (9–26) were enrolled. Functional fibrinogen maximum amplitude (FF MA) and FIBTEM maximum clot firmness (MCF) had identical correlation coefficients when compared with those of Clauss fibrinogen (both ρ = 0.64, P < 0.001), and FF MA and FIBTEM MCF correlated with each other (ρ = 0.71, P < 0.001). By logistic regression, the following cutoffs of fibrinogen levels were associated with increased odds of receiving a transfusion, red blood cell concentrates: Clauss <2.5 g/L, FF MA <14.9 mm, FIBTEM MCF <10 mm; fresh frozen plasma and platelets: Clauss <2.5 g/L, FF MA <16.9 mm, FIBTEM MCF <14 mm. ConclusionsThe viscoelastic hemostatic assays for determining fibrinogen levels, FIBTEM and FF, are both correlated with the Clauss fibrinogen level, and there are no differences in the strength of these correlations. In this study, specific fibrinogen levels at arrival to the emergency department were indicative, although not necessarily causal, of increased odds of receiving a transfusion.

Fibrin-based clot formation as an early and rapid biomarker for progression of postpartum hemorrhage: a prospective study

AbstractThis prospective, observational study investigated the utility of Fibtem A5 and Clauss fibrinogen as predictors of progression of postpartum hemorrhage (PPH). A consecutive cohort of 356 women experiencing 1000 to 1500 mL PPH was recruited. Fibtem and fibrinogen were measured and subsequent transfusions, invasive procedures, and bleed volume recorded. Women progressing to 8 U blood products (red blood cells [RBCs] + fresh frozen plasma [FFP] + platelets) had a median (interquartile range) fibrinogen and Fibtem A5 of 2.1 (1.8-3.4) g/L and 12 (7-17) mm, respectively, compared with 3.9 (3.2-4.5) and 19 (17-23) for those not progressing. On multivariate analysis, Fibtem was an independent predictor for progression to bleeds >2500 mL (95% confidence interval [CI], 0.85 [0.77-0.95]). Receiver operating characteristic area under the curve (95% CI) for progression to RBC transfusion was 0.67 (0.60-0.74) for fibrinogen and 0.61 (0.54-0.68) for Fibtem, and progression to >2500 mL was 0.71 (0.61-0.81) and 0.75 (0.66-0.85) for fibrinogen and Fibtem, respectively. Fibtem A5 <10 mm was associated with more prolonged bleeds (median [95% CI], 127 [44-210] compared with 65 [59-71] minutes; P = .018) and longer stay in the high-dependency unit (23.5 [18.4-28.5] compared with 10.8 [9.7-11.8] hours). Fibtem is a rapidly available early biomarker for progression of PPH.

Dabigatran assessment in patients with acute complications using routine coagulation assays

Complications while on dabigatran therapy, particularly bleeding and thrombosis, are occurring, and require laboratory assessment. The utility of routine coagulation assays has been previously evaluated in stable patients, but not those with acute complications. The purpose of this study was to determine how to employ routine coagulation assays to assess dabigatran in patients with acute complications. Seventeen patients on dabigatran presenting with various complications were evaluated. In addition, plasma samples with various fibrinogen levels were spiked in vitro with dabigatran ranging from low trough levels to the highest supratherapeutic concentrations reported (5000 ng/ml). INR, partial thromboplastin time (PTT), thrombin time (TT, Diagnostica Stago reagent), and fibrinogen were assayed and results compared to that of the Hemoclot Thrombin Inhibitors assay. Interference in the Clauss fibrinogen assay was assessed using a variety of commercial reagents. The majority of patients on dabigatran with acute complications demonstrated a significant negative bias in PTT results compared to normal plasma. TT remained highly sensitive to the presence of dabigatran (at least 10 ng/ml) under all circumstances investigated. There was wide variation in the sensitivity of commercial fibrinogen assays to dabigatran, with some even showing interference in the therapeutic range but this could be mitigated. The PTT is unreliable as a method for assessment of dabigatran in patients with acute complications. The TT assay is a simple and reliable alternative, particularly when combined with a fibrinogen level.

Fibrinogen and platelet contributions to clot formation

Thromboelastography (TEG) is used to diagnose perturbations in clot formation and lysis that are characteristic of acute traumatic coagulopathy. With novel functional fibrinogen (FF) TEG, fibrin- and platelet-based contributions to clot formation can be elucidated to tailor resuscitation and thromboprophylaxis. We sought to describe the longitudinal contributions of fibrinogen and platelets to clot strength after injury, hypothesizing that low levels of FF and a low contribution of fibrinogen to clot strength on admission would be associated with coagulopathy, increased transfusion requirements, and worse outcomes. A total of 603 longitudinal plasma samples were prospectively collected from 251 critically injured patients at a single Level 1 trauma center from 0 hour to 120 hours. TEG maximal amplitude (MA), FF MA, FF levels, von Clauss fibrinogen, and standard coagulation measures were performed in parallel. Percentage contributions of FF (%MA(FF)) and platelets (%MA(platelets)) were calculated as each MA divided by overall kaolin TEG MA. Coagulopathic patients (international normalized ratio ≥ 1.3) had significantly lower admission %MA(FF) than noncoagulopathic patients (24.7% vs. 31.2%, p < 0.05). Patients requiring plasma transfusion had a significantly lower admission %MA(FF) (26.6% vs. 30.6%, p < 0.05). Higher admission %MA(FF) was predictive of reduced mortality (hazard ratio, 0.815, p < 0.001). %MA(platelets) was higher than %MA(FF) at all time points, decreased over time, and stabilized at 72 hours (69.4% at 0 hour, 56.2% at 72 hours). In contrast, %MA(FF) increased over time and stabilized at 72 hours (30.6% at 0 hour, 43.8% at 72 hours). FF TEG affords differentiation of fibrin- versus platelet-based clot dynamics. Coagulopathy and plasma transfusion were associated with a lower %MA(FF). Despite this importance of fibrinogen, platelets had a greater contribution to clot strength at all time points after injury. This suggests that attention to these relative contributions should guide resuscitation and thromboprophylaxis and that antiplatelet therapy may be of underrecognized importance to thromboprophylaxis after trauma. Prognostic study, level III.

FUNCTIONAL FIBRINOGEN ASSAY INDICATES THAT FIBRINOGEN IS CRITICAL IN CORRECTING ABNORMAL CLOT STRENGTH FOLLOWING TRAUMA

Thromboelastography (TEG) is emerging as the standard in the management of acute coagulopathies in injured patients. Although TEG is sensitive in detecting abnormalities in clot strength, one shortcoming is differentiating between fibrinogen and platelet contributions to clot integrity. Current American algorithms suggest platelet transfusion, whereas European guidelines suggest fibrinogen concentrates for correcting low clot strength. Therefore, we hypothesized that a TEG-based functional fibrinogen (FF) assay would assess the contribution of fibrinogen and platelets to clot strength and provide insight to transfusion priorities. Blood samples were obtained from trauma patients on arrival to the emergency department or who were admitted to the surgical intensive care unit (n = 68). Citrated kaolin TEG, FF, and von Clauss fibrinogen levels (plasma-based clinical standard) were measured. Correlations were assessed using linear regression models. In vitro studies were also performed with adding fibrinogen concentrates to blood collected from healthy volunteers (n = 10). Functional fibrinogen and citrated kaolin TEG parameters were measured. Functional fibrinogen strongly correlated with von Clauss fibrinogen levels (R = 0.87) and clot strength (R = 0.80). The mean fibrinogen contribution to clot strength was 30%; however, there was a direct linear relationship with fibrinogen level and percent fibrinogen contribution to clot strength (R = 0.83). Traditional TEG parameters associated with fibrinogen activity (α angle and kinetic time) had significantly lower correlations with FF (R = 0.70 and 0.35). Furthermore, platelet count had only a moderate correlation to clot strength (R = 0.51). The addition of fibrinogen concentrate in in vitro studies increased clot strength (MA) (60.44 ± 1.48 to 68.12 ± 1.39) and percent fibrinogen contribution to clot strength (23.8% ± 1.8% to 37.7% ± 2.5%). Functional fibrinogen can be performed rapidly with TEG and correlates well with the standard von Clauss fibrinogen assay. Both fibrinogen and platelet contribution of clot strength can be derived from FF. Moreover, FF had a stronger correlation to clot strength, and increased levels were directly associated with increased percent contribution to clot strength. In vitro studies also demonstrated an increase in FF, clot strength, and percent fibrinogen contribution to clot strength with the addition of fibrinogen concentrate. These data suggest that fibrinogen should be addressed early in trauma patients manifesting acute coagulopathy of trauma.

Effects of Dabigatran and Rivaroxaban On Routine and Specialized Coagulation Assays: A Study Using Actual Patient Plasma Samples

Abstract 23 Background:To date, all published studies assessing the effects of the new oral anticoagulants dabigatran (dabi) and rivaroxaban (riva) on coagulation assays have used normal pooled plasma spiked with known concentrations of anticoagulant and therefore do not reflect the variability expected from patients taking these medications. Methods:We collected citrated venous blood samples from patients taking either dabi (n=43) or riva (n=10) for management of either atrial fibrillation or acute venous thrombosis. All subjects were required to have taken at least 5 doses of drug in order to reflect steady-state conditions and informed consent was obtained. The interval between last dose and phlebotomy ranged from 1 – 18 hours. Platelet-poor plasma was prepared and using the STA-R.. coagulometer (Diagnostica Stago), the following assays were performed: PT/INR using Neoplastin Plus.. (NP) and Innovin.. (IN), aPTT (Actin.. FS), thrombin time (TT), clot-based protein S (PS) activity, free PS antigen, chromogenic protein C (PC) activity, clot-based antithrombin activity, Clauss fibrinogen, dilute Russell’s viper venom time (DRVVT), and factor VIII (FVIII) activity at several dilutions (1:10 to 1:160). Hemoclot.. dilute thrombin time and STA.. -Rotachrom.. anti-Xa assays were performed for quantitation of dabi and riva drug levels, respectively, using Aniara commercial plasma calibrators. Results:Median INR levels for patients on dabi were 1.1 (IN) and 1.2 (NP) and for riva were 1.15 (IN) and 1.3 (NP). The proportion of subjects with INR levels above 1.3 for dabi were 15% (IN) and 28% (NP) and for riva were 0% (IN) and 40% (NP). Median aPTT levels were 42s dabi and 32.5s riva. Although 73% of patients on dabi had an elevated aPTT (>38s), 60% of these were only mildly elevated (39–45s). Only one subject on riva had an elevated aPTT. The TT was extremely sensitive to the presence of dabi, as 100% of treated subjects had an elevated TT (>20s) and 73% were above the linearity cutoff (>100s). All subjects on riva had normal TT. As shown in Figure 1A, the clot-based PS assay was affected markedly by dabi, with patients showing artificially high PS activity with increasing drug levels, but no such effect was observed for riva (Figure 1B). For both drugs, the PS free antigen and chromogenic PC assays were unaffected. Figure 1C shows dabi also caused false elevations in antithrombin activity, though it did not affect the Clauss fibrinogen assay, which uses a much higher concentration of thrombin. Riva had no discernable effect on the antithrombin and fibrinogen assays (data not shown). Figure 1D shows that increasing levels of both anticoagulants caused artefactual decreases in FVIII activity. Increasing dilutions of patient plasma caused progressive elevation of FVIII activity, with median activity levels nearly doubling between 1:10 and 1:160 dilutions for both dabi (0.65 to 1.28) and riva (0.81 to 1.41). Finally, the DRVVT assay was prolonged (>20% elevated versus normal plasma) for 86% of dabi and 60% of riva patient plasmas. No differences were noted for any of these results between dabi 110 mg (n=14) and 150 mg (n=29) dosing. Conclusions:For the assay platforms assessed in this study, the majority of patients taking dabi and riva had near-normal INR and aPTT levels. This is discrepant with results from studies using spiked plasma samples. Clinicians should be cautioned that normal INR and aPTT results do not rule out the presence of therapeutic anticoagulant levels and the results should not be used to guide clinical decisions. Clotting-based thrombophilia testing should be avoided for patients taking these medications due to assay interferences, particularly with dabigatran. [Display omitted] Disclosures:Teal:Bayer: Consultancy, Honoraria; Boehringer-Ingelheim: Consultancy, Honoraria. Lee:Bayer: Honoraria; Boehringer-Ingelheim: Honoraria.

Clinical phenotype, laboratory features and genotype of 35 patients with heritable dysfibrinogenaemia.

Heritable dysfibrinogenaemia (HD) is a rare qualitative disorder of fibrinogen (FGN). To better describe the clinical, laboratory and genotypic spectrum of HD, we evaluated 35 subjects identified at two UK centres using laboratory criteria. 12/35(34%) subjects with HD experienced bleeding (bleeding score >1 at any site), 3/35(9%) thrombosis and 20/35(57%) were asymptomatic. Amongst subjects with bleeding, symptoms were typically mild, at one anatomical site and seldom occurred after invasive procedures. All subject showed dry clot weight within or above laboratory reference interval (median 3·2 g/l; range 1·9-5·1), reduced Clauss fibrinogen (median 0·52 g/l; range 0·21-1·3), and prolonged thrombin (median 30·7 s; range 21·3-45·7) and reptilase (median 42·0 s; range 20·0-68·0) times. In all subjects, the prothrombin time ratio (PTR), determined by Sysmex CA-1500 coagulometer and Innovin activator, was abnormal (median 1·42; range 1·22-1·61). The activated partial thromboplastin time ratio and PTR with other coagulometers and activators were comparatively insensitive to HD. All subjects with HD harboured heterozygous candidate nucleotide variations within known hotspots in the FGN genes. The HD variants identified in this cross-sectional study seldom have significant clinical manifestations and show similar laboratory features irrespective of genotype. Selection of coagulometer and PT activator may markedly affect the detection of new HD cases using coagulation screening tests.

664 COMBINATION OF BANDING LIGATION AND MEDICAL INTERVENTION FOR SECONDARY PREVENTION IN ESOPHAGEAL VARICES: A SYSTEMATIC REVIEW AND META-ANALYSIS OF RANDOMIZED TRIALS

Introduction: Elevation of Prothrombin time (PT) and/or INR following Hepatectomy is a well-known phenomenon and can lead to delays in initiation of thrombo-prophylaxis and epidural catheter removal with administration of prophylactic transfusions of Fresh Frozen Plasma (FFP). Recently, the role of PT-INR has been challenged as this test does not reflect the balance between proand anti-coagulants that is often preserved. Aims: We sought to compare these conventional tests with more global assays of hemostasis such as ROTEMTM, Thrombin Generation and Coagulant Factor activity. Design: We carried out a prospective observational study with full ethical approval in 48 patients undergoing major hepatectomy (>30% resection). Methods: Conventional tests, ROTEM analysis, endogenous thrombin potential (ETP) and proand anti-coagulant activity were measured at baseline, immediately after resection and postoperative days (POD) 1, 2, and 5. Results: Values are mean (±SD). Mean INR peaked at 1.8 (±0.42) on POD 1 and returned to baseline by POD 5. Platelets reached a nadir of 163 (±78) on POD 1 and exceeded baseline by POD 5. Clauss Fibrinogen stayed within normal range but peaked on POD5 at 4.4 g/dL (±1.4). ROTEM EXTEM and INTEM parameters as well as mean ETP values all remained within normal range for the entire study period. Mean levels of factor II, VII, IX and X all stayed above 30iu/dl for the entire study period. Factors VIII and von Willebrand factor (vWf) increased steadily from POD1 onwards. Mean levels of Protein C, Protein S and Anti-Thrombin all dropped to below 50iu/dl by POD1 but only protein S recovered by POD 5. Conclusions: Although PT/INR increased following major hepatic resection, ETP and viscoelastic parameters remained within normal limits and procoagulant factor levels were above the critical threshold of 30% required for normal haemostasis. Levels of endogenous anti-coagulants drop post-operatively and apart from protein S, remain suppressed at POD5. This combined with supranormal levels of factor VIII, vWf may create a hypercoaguable state. This has important healthcare implications if patients are not receiving thromboprophylaxis based on the INR, with inappropriate FFP transfusions and delays in invasive procedures.

The Impact of Hematocrit on Fibrin Clot Formation Assessed by Rotational Thromboelastometry

Rotational thromboelastometry (ROTEM®)-based FIBTEM is used perioperatively to assess the extent of fibrin polymerization in whole blood. In FIBTEM, cytochalasin D eliminates the contribution of platelets to whole blood clotting, but changing levels in fibrin(ogen) and erythrocytes may differently affect clot formation. Because dynamic changes of hematocrit are not reflected in plasma fibrinogen measurements, we hypothesized that the lack of erythrocytes in isolated plasma measurements would affect the relationship between the Clauss method and whole blood-based FIBTEM during cardiac surgery. Therefore, in the current study we investigated the influence of perioperative hematocrit changes on FIBTEM and fibrinogen measurements. Blood samples were collected from 6 consenting healthy volunteers. FIBTEM tests were run before and after serial in vitro dilutions of whole blood with saline or autologous plasma (5:1, 2:1, and 1:1 v/v). We then evaluated the relationship between FIBTEM-maximal clot firmness (MCF) and the Clauss fibrinogen method in relation to hematocrit values before and after cardiac surgery. Pearson correlation coefficients were determined between laboratory test results and ROTEM variables. Upon in vitro hematocrit reduction, FIBTEM-MCF was progressively decreased depending on the extent of saline dilution, but it was increased by 31% after 1:1 volume replacement with autologous plasma (P < 0.05). In samples from cardiac patients (150 measurements in 50 patients), the overall correlation coefficient between FIBTEM-MCF and plasma fibrinogen was 0.80 (P < 0.001). In hemodiluted blood samples (during surgery or at intensive care unit), FIBTEM-MCF 10 mm corresponded to plasma fibrinogen levels of 200 mg/dL. In the subgroup analysis (n = 50 each), according to hematocrit levels (<25%, ≥25% to 30%, ≥30%), plasma fibrinogen levels of 200 mg/dL corresponded to 11 mm, 10 mm, and 8 mm of FIBTEM-MCF, respectively. The correlation between FIBTEM-MCF and plasma fibrinogen was higher at lower hematocrit (<25%) than at higher hematocrit (>30%) (r = 0.88 and 0.67, respectively). Perioperative changes in hematocrit affect the correlation between plasma fibrinogen levels and FIBTEM-MCF values. The higher correlation between FIBTEM-MCF and plasma fibrinogen with lower hematocrit (<25%) indicates that FIBTEM is a practical method to determine the need for fibrinogen replacement in bleeding patients who typically develop perioperative anemia.

Quantitative reagent-free detection of fibrinogen levels in human blood plasma using Raman spectroscopy

Fibrinogen assays are commonly used as part of clinical screening tests to investigate haemorrhagic states, for detection of disseminated intravascular coagulation and as a predictor of a variety of cardiovascular events. The Clauss assay, which measures thrombin clotting time, is the most commonly used method for measuring fibrinogen levels. Nevertheless, inconsistencies are present in inter-manufacturer reagent sources, calibration standards and methodologies. Automated coagulation analysers, which measure changes in optical density during the prothrombin time (PT-Fg), have found use in many hospitals. However, the PT-Fg method is found to give falsely elevated values due to varying choices of calibrants, reagents and analysers. As an alternative, Raman spectroscopy has previously been applied to the analysis of blood and its various constituents to determine various analyte concentrations such as glucose, urea, triglycerides and cholesterol. In this study, Raman spectroscopy was investigated for its ability to accurately quantify fibrinogen concentration in blood plasma. Samples collected from 34 patients were analysed by Raman spectroscopy and the resultant spectra were fitted with a Partial Least Squares Regression model using target values obtained through a pre-calibrated Clauss fibrinogen assay. Various spectral pre-processing methods were utilised to prepare data to be entered into a calibration model. A root mean square error of prediction of 0.72 ± 0.05 g/L was achieved with as few as 25 spectra. In this pilot study, Raman spectroscopy has been demonstrated to be a robust technique providing rapid and reagent-free quantification of fibrinogen levels in blood plasma and a potential alternative to the Clauss assay.

Phenotype and Genotype of 22 Subjects with Heritable Dysfibrinogenaemia

Abstract 1212▪▪This icon denotes a clinically relevant abstractThe heritable dysfibrinogenaemias (HD) are a heterogenous group of qualitative disorders of fibrinogen in which there is abnormal fibrin clot formation. Affected individuals usually display prolonged thrombin (TCT) and reptilase clotting times (RCT) and reduced plasma fibrinogen activity. The plasma fibrinogen concentration is normal or is reduced less than the fibrinogen activity. HD is associated with pathogenic mutations in FGA, FGB or FGG genes that encode the fibrinogen Aα, Bβ and g chains respectively. However, most clinical descriptions of HD comprise single case reports and the range of phenotype and genotype in the wider population is difficult to estimate. In order to improve understanding of this disorder, we now report a cross-sectional survey of 22 subjects with HD identified at 2 UK regional Haemostasis centres from 2001–11.We recruited index cases from our Haemostasis laboratory referral practice who satisfied all the following criteria- 1) prolonged TCT; 2) prolonged TCT with protamine (TCTP) and/or prolonged RCT; 3) reduced Clauss fibrinogen and 4) normal fibrinogen dry clot weight. Demographic and clinical characteristics were recorded using a standardised case report form. Genetic analysis was performed by Sanger sequencing of FGA exon 2, FGB exon 2 and FGG exon 8 which we identified as mutation hotspots from the fibrinogen mutation database (http://www.geht.org/databaseang/fibrinogen/). Phenotype and genotype was also determined in available first degree family members.We identified 16 index cases and 6 family members with HD (median age 41, range 12–78; 9 males). HD was identified in plasma submitted to our Haemostasis laboratories for the investigation of bleeding in 8/16 (50%) index cases, for stroke in 1/16 (6%) and for ‘routine coagulation screening' in 7/16 (44%). Mild mucocutaneous bleeding or menorrhagia was documented in the clinical records of 13/22 (59%) subjects and thrombotic stroke in 1/22 (5%). 8/22 (36%) subjects were asymptomatic.The prothrombin time (PT) was prolonged in 21/22 (95%) subjects (median 15.3 s; range 11.4–16.7; reference interval (RI) 9.6–11.6) but the activated partial thromboplastin time (aPTT) was prolonged in only 1/22 (5%; median 23.8 s; range 22.8–37.0, RI 24.0–32.0). The median TCT was 28.6 s (range 20.6–45.7; RI 13–19) and TCTP 31.9 s (n=11; range 20.0–35.5; RI 13–19). The median RCT was 37s (n=12; range 19–68; RI 15–19), Clauss fibrinogen 0.49 g/dL (range 0.29–1.3; RI 1.8–3.6) and dry clot weight 2.3 g/dL (range 1.9–4.2; RI 1.8–3.6). Heterozygous missense nucleotide variations were identified in the candidate exons of the fibrinogen genes in all 16/16 (100%) index cases and segregated with the HD phenotype in all available family members. These variations predicted aminoacid substitutions at the fibrinopeptide A cleavage site in the fibrinogen Aa chain in 10/16 (63%) index cases (FGA p.R35H (n=4); p.R35S (n=3); p.R35P (n=2) and p.G36S (n=1); numbered from RefSeq NP_000499.1) or at nearby residues (FGA p.V39D (n=1) and p.F27C (n=1)). FGG variations were identified in 3 index cases that predicted aminoacid substitutions in the fibrinogen g chain region necessary for D:D domain interactions during fibrin monomer assembly (FGG p.R301H (n=1), p.D342G (n=1) and p.A367T (n=1); RefSeq NP_000499.1). One subject harboured a FGB variation that predicted a p.R44C substitution (Refseq NP_005132.2) at the fibrinopeptide B cleavage site in the fibrinogen Bb chain. The FGA p.R35S, p.R35H, FGB p.R44C and FGG p.R301H and p.A367T substitutions have been associated previously with HD.We show that the HD laboratory phenotype is rare and was frequently identified in subjects with mild bleeding. However, HD was also commonly asymptomatic and did not segregate with bleeding symptoms within affected families suggesting that the HD variants identified in this study do not confer significant bleeding risk. The prevalence of mild bleeding in some study subjects may reflect recruitment bias. We also show that HD is strongly associated with nucleotide variations in the FGA, FGB or FGG genes. All the observed nucleotide variations have been previously associated with HD and/or predict aminoacid substitutions in critical regions for normal fibrin polymerization. Defective fibrin polymerization may affect endpoint determination in some automated clotting assays and is likely to account for the consistent finding of prolonged PT in the study subjects. Disclosures:No relevant conflicts of interest to declare.

Correlation of a Condensed Bleeding Score with New Diagnosis of a Congenital Bleeding Disorder in Patients Referred to a Tertiary Centre

Abstract 1226▪▪This icon denotes a clinically relevant abstractThe performance and utility of a condensed bleeding score (Bowman et al, J Thromb Haemost., 2008;6:2062) in relation to the diagnosis of a congenital bleeding disorder in new referrals to a regional haemostasis clinic over an 8 month period is presented. Between November 2010 and June 2011, 50 patients over the age of 16 (median age, 31 years; range, 16–79), including 32 females, were referred for investigation of a possible congenital bleeding disorder following detection of abnormal coagulation results and/or presentation with a bleeding history. A bleeding score was performed as part of their initial assessment. 12(24%) patients were from local referral and 38(76%) patients were referred from other hospitals in the region for further investigation of a suspected bleeding disorder. Basic coagulation tests (activated partial thromboplastin time (APTT), prothrombin time Clauss fibrinogen and platelet count) were normal in the referred patients from other centres. 50% (6/12) of the local referrals were for investigation of a prolonged APTT detected on routine coagulation screening prior to major surgery.The median bleeding score was 6 with a range of −1 to 14 (Table 1). The presence of a congenital bleeding disorder was confirmed in 31 of the 50 patients (62%), including 19/31 (61%) of the female patients and 12/31(39%) of the males. Correlation of an abnormal bleeding score (score ≥ 4) with diagnosis of a congenital bleeding disorder was only seen for diagnosis of type 1 Von Willebrand Disease (VWD) (Table 2). Analysis of the cases with low scores and abnormal results identified two groups of patients; firstly, those who had not yet had a significant haemostatic challenge, and secondly, those in whom the abnormal coagulation results were explained by a non-haemostatically significant reduction in a coagulation factor level (e.g. FVII, 15%; dysfibrinogenaemia; F XII deficiency). These clinically insignificant laboratory abnormalities explain the discrepancy between the number of patients with abnormal laboratory tests (35) and the number of patients diagnosed with a congenital bleeding disorder (31).Table 1Bleeding score (range)Number of patients with normal lab resultsNumber of patients with abnormal lab results−1 to +1382–44105–74128–102311–1422Total1535Table 2DiagnosisNumber of patientsMedian bleeding scoreAge rangeType 1 VWD116 (4–10)17–51Type 2 VWD48 (5–13)17–36Factor XI123 (1–8)17–76Platelet function defect46 (2–9)17–57Compared to previous reports the range of scores found with this assessment tool was narrow and could not exclude patients from further laboratory assessment. However the condensed bleeding score has only been validated prospectively for the diagnosis of type 1 VWD and all patients in this cohort who were diagnosed with type 1 VWD had an abnormal bleeding score (≥ 4). This observation supports the role of this scoring system in the assessment of patients for type 1 VWD. The use of the condensed bleeding score in assessing patients with suspected factor XI deficiency is difficult due to the lack of a phenotypic relationship between residual factor XI activity and a bleeding tendency. Furthermore, although factor XI deficiency is a rare congenital bleeding disorder in our cohort of patients 12/31(39%) were diagnosed with factor XI deficiency. This may explain the overall lack of correlation between bleeding score and diagnosis of a congenital bleeding disorder. Patients who have an abnormal bleeding score but normal laboratory tests need consideration of further investigations before concluding they are normal. The possibility of an acquired bleeding disorder should be considered. A thorough drug history is also important as one of the patients with a bleeding score of 14 was taking a non-steroidal anti-inflammatory drug.The use of the condensed bleeding score in the detection of congenital bleeding disorders other than type 1 VWD requires further validation in a larger number of patients. Disclosures:No relevant conflicts of interest to declare.

A Novel Missense Mutation in FGG (c.944C>A) Encodes for An Amino Acid Change (p.Ala315Asp) in the Gamma Chain of Fibrinogen Causing Hypofibrinogenemia and a Thrombotic Phenotype

Abstract 856Fibrinogen plays a central role in maintaining hemostasis. Disruption of its normal function may lead to hemorrhagic or thrombotic events. Fibrinogen is encoded by three genes, FGA, FGB, and FGG, clustered on chromosome 4q28-q31. Hereditary defects of fibrinogen, although uncommon, can affect the quantity (hypofibrinogenemia and afibrinogenemia) or the quality (dysfibrinogenemia) of the circulating protein. We report a family with two affected individuals (father and daughter) that presented with a mild bleeding predisposition, but also exhibited thrombotic events, i.e. unprovoked deep vein thrombosis in a father and an in utero middle cerebral artery stroke in a daughter. The Clauss fibrinogen levels for the father and daughter were 105 and 98 mg/dL (normal 150–400 mg/dL) and fibrinogen antigen levels were 138 and 152 mg/dL (normal 170–400 mg/dL), respectively. Thrombin times were prolonged in both patients. All other coagulation factors were within normal range and the patients did not carry either the prothrombin G20210A or factor V Leiden mutation.All exons and intron-exon junctions of the three fibrinogen genes were sequenced. The father and daughter were found to be heterozygous for a novel missense mutation in the gamma chain (Ala315Asp) while this mutation was not observed in an unaffected family member (mother of the daughter).Transient transfection experiments using CHO-K1 cells showed that gamma chain with p.Ala315Asp mutation was detected in both cell lysates and supernatant, although in reduced amount as compared with cells transfected with wild-type (WT) FGG cDNA.Structurally, there was no significant difference in fibrin fiber diameter, as measured manually by scanning electron microscopy, between the affected (father and daughter), and the daughter's unaffected mother. Similarly, there was no difference in thrombus height between the three individuals. As expected, hydraulic permeability of platelet rich plasma clots correlated with the fibrinogen levels in each person. Clotting kinetics in plasma of affected family members was consistent with hypofibrinogenemia, but did not suggest abnormal fibrin polymerization. Clot structure and fibrin deposition was further characterized with a microfluidics flow assay, an experimental technique in which platelet and fibrin accumulation is observed in real-time using fluorescent probes in whole blood under shear stress on a collagen/tissue factor micropatterned surface. Interestingly, fibrin density and platelet accumulation, as measured by platelet and fibrin surface area coverage, was highest in the father and daughter, the two individuals with hypofibrinogenemia and thrombotic phenotype.In order to evaluate the possible changes resulting from the Ala315Asp mutation we examined predicted alterations in protein structure, energy, and protein-protein interaction between two fibrinogen molecules via in silico molecular modeling using published protein crystal structure. The molecular modeling showed that alanine for aspartic acid substitution predicted to result in substantial changes in overall conformation of the interaction interface (distal D-domain) between the two fibrinogen molecules due to introduction of a bulkier side chain. Potential energy (energy of minimization) for Ala315Asp mutant homodimer, and for WT-mutant heterodimer were lower than the WT homodimer, predicting a more stable structure from an energetic standpoint. Overall interaction energy (measurement of the energy of association between the two molecules) was lower for Ala315Asp mutant homodimer as compared with WT homodimer or WT-mutant heterodimer, suggesting more favorable interaction for Ala315Asp mutant dimers. Overall, the in silico modeling studies predicted that Ala315Asp mutation may lead to more rapid or stronger interaction between fibrinogen molecules.These data suggest that a novel FGG mutation, c.944C>A, that predicts pAla315Asp change, if expressed and incorporated into fibrinogen molecule in vivo, may result in a more stable or more resistant to lysis clot. Characterizing the association between a specific genetic defect and its phenotypic expression will advance the current understanding of the molecular and biochemical mechanisms of the inherited disorders of fibrinogen, and may contribute to the development of safer therapeutic interventions for the patients with fibrinogen disorders. Disclosures:No relevant conflicts of interest to declare.

Clauss assay and fibrinogen protein estimated by capillary zone electrophoresis

The clottability and the amount of total protein in fibrinogen provide information about qualitative or quantitative alterations. We aimed to evaluate whether capillary zone electrophoresis (CZE) Capillarys II analyzer with the protein 6 buffer is able to estimate the amount of fibrinogen antigen. Citrated plasmas were assayed for clottable fibrinogen, and any relationship with the β(2)-globulin fraction (percentage of the area under the curve) was evaluated. The integration method used was "tangent skimming" in order to reduce the overestimation due to high protein background. Linearity was optimized according to the ICH Q2R1 recommendations and evaluated using polynomial regression. The precision was computed in accordance with the Clinical and Laboratory Standards Institute EP5-A2 protocol. In patients, clottable fibrinogen (Clauss method) was compared to its protein CZE amount by Passing and Bablok regression and the Bland-Altman plot. The correlation was linear y=0.0744+0.8991x (R(2)=0.9707) within the range of 2.26-17.26 μmol/L. The repeatability and the within-device precision were <15% for three levels of percentage of the β(2)-globulin fraction (1.61%, 3.51%, and 9.24%). In patients, clottable fibrinogen and its protein amount were similar (-0.1779+0.9654x). The ratio activity/protein was 1.08 ± 0.32 (mean ± 2 SD). CZE with the Capillarys II and the buffer protein 6 is an easy method. It is a good candidate for estimation of the concentration of fibrinogen antigen, which may have diagnostic utility for the screening of quantitative or qualitative fibrinogen abnormalities.

Identifying Patients at High Risk for Deep Vein Thrombosis (DVT) After Acute Stroke Using Clinical and Laboratory Predictors.

AbstractAbstract 3825The national Venous Thromboembolism prevention programme in England emphasises reducing hospital-associated thrombosis by ensuring adequate prophylaxis, but this is problematic in stroke patients due to poor uptake of pharmacological prophylaxis and lack of evidence for mechanical measures. Our prospective study aimed to develop clinical and laboratory predictors of thrombosis in patients after acute stroke. Participants were recruited within 48 hours of admission to the Acute Stroke Centre at King's College Hospital London UK, between June 2009 and June 2010. 142 participants were recruited with complete data available from 86 participants. Racial distribution was as follows: 75% Caucasian, 19% African/Caribbean, 6% Asian. Mean age was 69 years (SD15) and 45/86 (52%) were female. 73 strokes were ischaemic and 13 haemorrhagic. In the ischaemic stroke group, 33 of 73 (45%) were thrombolysed as per National stroke strategy. Baseline demographic data and clinical evaluation using the NIH Stroke Scale (NIHSS) and Barthel Index (BI) were recorded. Analysis of prothrombotic markers was performed including D-dimer (DD), Clauss fibrinogen (CFIB) and thrombin generation (TG) measured by calibrated automated thrombography (CAT). The TG assay measured kinetics in platelet poor plasma including lag time (LT) - time taken to generate 10nM of thrombin; time to peak (TTP) - time in reach maximum TG; peak thrombin generation (PTG)- maximum thrombin concentration; and endogenous thrombin potential (ETP) - area under the curve (expressed in nmol/L of thrombin). In the second week all laboratory tests were repeated and lower limb compression ultrasound scan (CUSS) performed. 18/86 (20.9%) had objectively confirmed DVT (5 proximal and 13 distal). 4 DVTs (2 proximal and 2 distal) occurred in patients with haemorrhagic stroke, and 14 DVTs (3 proximal and 11 distal) occurred in patients with ischaemic stroke. Both BI (p=0.02) and body mass index (p=0.05) but not NIHSS (p=0.13) were associated with DVT. TG assay parameters and CFIB were not associated with the risk of DVT. Elevated DD was significantly associated with DVT development both at baseline (DD 2425 ng/ml vs 1010 ng/ml, p=0.0010) and at week 2 (DD 2770 ng/ml vs 830 ng/ml, P<0.001). A subsequent analysis examining the effects of NIHSS, BI and DD using multiple logistic regression, indicated that only DD at baseline was significantly associated with DVT. After adjusting for the effect of this variable, no other variables were found to be significant. ROC curves were used to determine the optimum cut-off for each of the three key variables (NIHSS, BI and DD). The results indicated that the area under the ROC curve (AUC) was greatest for DD (0.77), followed by BI (0.70), and NIHSS (0.62). The best predictive performance for DVT was for DD, with the cut-off of 1230 ng/ml providing a sensitivity of 88% and a specificity of 64%. Limitations of the analysis include the relatively small number of DVT cases and further patients will be accrued before the study is complete. In conclusion, the incidence of DVT after stroke remains high (20.9%) despite the trend towards more aggressive treatment with thrombolysis. BI and DD (but not NIHSS, TG and CFIB) are important predictors of DVT in this population, and are of potential use in identifying high-risk patients for thromboprophylaxis following acute stroke. Disclosures:No relevant conflicts of interest to declare.

Coagulopathy in a patient with nephrotic syndrome

A 55-year-old Caucasian woman presented with a 5week history of increasing lower limb edema associated with a two-pound daily weight gain. She was feeling well otherwise; she denied shortness of breath, chest discomfort, recurrent headaches, myalgia, arthlagia, or constitutional symptoms. She had respiratory infection 4 months before, but the infection has resolved. The patient presented with peripheral edema. The differential diagnosis includes right-sided heart failure, liver cirrhosis, nephrotic syndrome, drug-induced edema, and lymphoedema. Her preceding upper respiratory tract infection suggests postinfectious cardiac or renal pathology. The patient did not have any prior bleeding episode. The medical history was significant for Graves’ disease treated with radioactive iodine ablation. The only medication was daily thyroid supplementation. She was married and had two uncomplicated pregnancies. She was a lifelong nonsmoker. She denied any alcohol intake or illicit drug use. She was a pharmacist without any known exposure to toxic chemicals. On examination her vital signs were normal. She was not in any distress. Examination of her legs revealed bilateral pitting peripheral edema up to the knee level. There was no jaundice, lymphadenopathy, or bleeding sign. Her cardiac examination was normal and her jugular venous pressure (JVP) was not elevated. Respiratory examination was normal with no crackles on auscultation. Abdominal examination revealed no palpable hepatosplenomegaly or ascites. The fact that this patient was not on any medication other than long term thyroid supplement makes druginduced edema unlikely. The finding of normal JVP on clinical examination is against the diagnosis of heart failure. Lymphedema is typically nonpitting. Thus, the most likely differential diagnosis includes nephrotic syndrome and early liver cirrhosis. Patient’s hemoglobin level was 19.0 g/dL (normal 11.5– 16.5 g/L), platelet count 305,000/mm (normal 150,000– 400,000/mm). The blood urea nitrogen was 14.4 mg/dL (normal 7.0–18.0 mg/dL), creatinine 0.93 mg/dL (normal <1.5 mg/dL), albumin 2.0 g/dL (normal 3.5–5.5 g/dL), cholesterol 587 mg/dL (normal <200 mg/dL), and triglyceride 447.1 mg/dL (normal <160 mg/dL). The results of liver function tests were normal. Urinalysis revealed 11 blood, 31 protein, and granular casts. Twenty-four hour urine protein excretion was 15,000 mg/d. The combination of heavy proteinuria, minimal hematuria, hypoalbuminemia, and hypercholesterolemia suggests nephrotic syndrome. Nephrotic syndrome may be associated with primary kidney disease, such as minimal change disease, focal segmental glomerulosclerosis, and membranous nephropathy. Systemic diseases including diabetes mellitus, systemic lupus erythematosus, amyloidosis, or Fabry’s disease also cause nephrotic syndrome. International normalized ratio (INR) was increased to 2.6 and activated partial thromboplastin time (aPTT) was prolonged to 68 s but thrombin clotting time was normal. Clauss fibrinogen level was slightly elevated at 480 mg/dL (normal range 200–400 mg/dL). D-dimer level was not elevated. In general, patients with nephrotic syndrome are in a hypercoagulable state because of urinary loss of antithrombin III and enhanced platelet reactivity, but this patient’s coagulation tests were paradoxically prolonged although there was no bleeding symptom. Simultaneous prolongation of both INR and aPTT suggests common coagulation pathway abnormalities like liver dysfunction, vitamin K deficiency, disseminated intravascular coagulopathy (DIC), primary fibrinolysis or post-thrombolytic therapy. However, the normal thrombin clotting time and borderline high fibrinogen level make abnormalities in the conversion pathway of fibrinogen to fibrin less likely. Normal fibrinogen and D-dimer level are atypical for DIC. A deficiency of multiple coagulation factors in both intrinsic and extrinsic coagulation pathways commonly occurs in vitamin K deficiency or hepatic dysfunction. However, 48 hr after administration of 5 mg oral vitamin K, the coagulation indices remained prolonged with an INR at 2.6 and aPTT at 63 s. To further define the causes of coagulopathy, clotting factor levels were assayed individually. Factor II level was high at 166%, factor VII 179%, but factor VIII:C was low at 7%, factor X 3%, whereas factor XII level was borderline low at 47%. (Normal range of these clotting factors was 50–150%). Although acquired factor XII deficiency has been reported in patients with nephrotic syndrome [1], deficiency of multiple coagulation factors suggests acquired conditions, either due to insufficient production or excessive consumption. Coagulation factors II, V, VII, IX, X, XI, and XII are synthesized in the liver, and in addition factor VIII also

Level of agreement between the Clauss fibrinogen test and the rotation thromboelastometry FIBTEM test in cardiothoracic surgery

Level of agreement between the Clauss fibrinogen test and the rotation thromboelastometry FIBTEM test in cardiothoracic surgery

New Fibrinogen Substitution (gSer313Arg) with Hypodysfibrinogenemia and the Use of Thromboelastography in Diagnosis of Hypo/Dysfibrinogenemia.

Abstract 4437 IntroductionFibrinogen, also known as factor I, is an integral component of both the coagulation and fibrinolytic pathways. The functions of this 340 kDa glycoprotein include fibrin clot formation, non-substrate thrombin binding and platelet aggregation. It is encoded by 3 genes, FGA, FGB and FGG, clustered on chromosome 4q. Disorders of fibrinogen are extremely rare, and may be qualitative or quantitative, resulting from defects in synthesis, processing, polymerization, crosslinking or clot lysis. Clinical manifestations vary from life threatening bleeding disorders to thromboembolism. We report here the identification of a novel g313Ser®Arg substitution in the gD domain that affects both expression level and fibrinogen function in a 3 year old African American child and her mother with a history of epistaxis and menorrhagia respectively. ResultsPlasma samples showed low Clauss fibrinogen concentrations (0.5 and 0.4 g/L) and extended thrombin clotting times (40 and 39 s) for both the proband and her mother (normal 1.5-4.0 g/L and 18-26 s respectively). Estimation of physical fibrinogen concentrations by HPLC quantitation of fibrinopeptides gave values of 1.2 and 1.5 g/L respectively (normal 1.5-4.0 g/L). While these latter values clearly indicate hypofibrinogenemia, the discrepancy between the activity based and physical concentrations suggest a concomitant functional defect. Thromboelastography (TEG) performed on the child showed a low angle and reduced maximum amplitude indicating a platelet or fibrinogen dysfunction or deficiency. A TEG® platelet mapping‘ study confirmed a significant impairment of fibrinogen function. Examination of purified fibrinogen by SDS-PAGE showed a normal pattern of Aa, Bb and g chains, with an additional minor band in the g region showing increased mobility. Electrospray ionisation mass spectrometry of separated chains again confirmed an abnormality in the g chain with measured masses of 48,399 and 48,400 Da from two separate measurements compared to control values of 48,394 Da (SEM 0.6, n=3). DNA sequencing of FGG revealed a novel heterozygous mutation of AGT to CGT at codon 339 (c.1015A>C). The g313Ser®Arg substitution has not been previously reported and we have named the new variant fibrinogen Detroit II. ConclusionsThe majority of variant chains are not secreted resulting in hypofibrinogenemia. However, a minor population of dysfunctional variant g chains (∼15 % of total g chains) was expressed in plasma fibrinogen causing hypodysfibrinogenemia. TEG was a useful assay for the diagnosis of the hypo/dysfibrinogenemia and should be considered in the evaluation of patients with bleeding symptoms. Disclosures:No relevant conflicts of interest to declare.

Influence of Different Hydroxyethyl Starch (HES) Formulations on Fibrinogen Measurement in HES-Diluted Plasma

Fibrinogen is the first coagulation factor becoming critical in dilution coagulopathy. Volume replacement in major blood loss is performed with large volumes of crystalloid and colloid solutions. The latter has been shown to compromise accurate photo-optical measurement of fibrinogen. This study determined the influence of different hydroxyethyl starch (HES) formulations. Citrated plasma samples of 8 healthy volunteers were diluted by 30% or 50% with either HES 10% (200/0.5; HES-200), HES 6% (70/0.5; HES-70), or HES 6% (450/0.7; HES-450). Fibrinogen concentrations were determined by photo-optical measurement (Behring coagulation system [BCS]: derived fibrinogen, or Clauss fibrinogen, calibrated for high [CLS] or low fibrinogen concentrations [CLS-low]) as well as mechanical end point determinations (KC4: CLS-KC4). Measured values were compared with calculated values. On average and across all photo-optical methods, fibrinogen concentrations were overestimated, particularly with HES-200. Hydroxyethyl starch-70 and HES-450 did not differ much from each other. Overestimation was relatively greater for 50% dilutions with all HES formulations. Surprisingly, overestimation was most prominent with CLS-low, the method supposed to most reliably measure low fibrinogen concentrations; overestimation amounted to 92% and 120% with HES-200, 54% and 73% with HES-70, and 51% and 79% with HES-450, for 30% and 50% dilutions, respectively. In contrast, CLS-KC4 always yielded sufficiently accurate results. The study showed that all HES solutions more or less impaired the fibrinogen measurement with the photo-optical method. In particular, overestimation with CLS-low may prevent timely fibrinogen replacement in major blood loss. Hydroxyethyl starch concentration appears to be more relevant for this effect than its molecular size.