Kirsten Christiansen

Whole blood coagulation thrombelastographic profiles employing minimal tissue factor activation

Summary.  We investigated whole blood coagulation by thrombelastography (TEG) employing activation with minute amounts of tissue factor (TF). Continuous raw data captured were transformed into novel parameters, such as the maximum velocity (MaxVel) and the time to maximum velocity (t,MaxVel) of whole blood clot formation. The courses of the whole blood clot development were very similar to thrombin generation curves reported in plasma. In this assay healthy women (n = 30) showed an earlier onset and an increased coagulation velocity compared to healthy men (n = 30). In patients with severe hemophilia, and persons undergoing thromboprophylaxis, distinctly abnormal coagulation profiles were observed with a decrease in the MaxVel, as well as a prolonged t,MaxVel. Changes appeared to be dependent on the nature and severity of the hemostatic deficit. Preliminary studies in patients substituted with recombinant factor VIIa demonstrated a marked change in the coagulation profile, in which the MaxVel and t,MaxVel shifted towards normal in a dose‐dependent way. Data suggest that the whole blood coagulation TEG profile, following activation with minute amounts of TF, may reflect the hemostatic potential in patients suspected of impaired hemostasis.

Diagnostic Performance and Therapeutic Consequence of Thromboelastometry Activated by Kaolin versus a Panel of Specific Reagents

Background:Thromboelastography/metry (TEG®; Haemoscope, Niles, IL/ROTEM®; Tem International GmbH, Munich, Germany) is increasingly used to guide transfusion therapy. This study investigated the diagnostic performance and therapeutic consequence of using kaolin-activated whole blood compared with a panel of specific TEM®-reagents to distinguish: dilutional coagulopathy, thrombocytopenia, hyperfibrinolysis, and heparinization. Methods:Blood was drawn from 11 healthy volunteers. Dilutional coagulopathy was generated by 50% dilution with hydroxyethyl starch 130/0.4 whereas thrombocytopenia (mean platelet count 20 ×109/l) was induced using a validated model. Hyperfibrinolysis and heparin contamination were generated by tissue plasminogen activator 2 nM and unfractionated heparin 0.1U/ml, respectively. Coagulation tests were run on ROTEM® delta. Results:Kaolin-activated whole blood showed no differences between dilutional coagulopathy and thrombocytopenia (mean clotting time 450 s vs. 516 s, &agr;-angle 47.1° vs. 41.5°, maximum clot firmness 35.0 mm vs. 34.2 mm, all P values ≥0.14). Hyperfibrinolysis specifically disclosed an increased maximum lysis (median: 100%, all P values less than 0.001), and heparin induced a distinctly prolonged clotting time (2283 s, all P values less than 0.02). The coagulopathies were readily distinguishable using a panel of TEM-reagents. In particular, dilutional coagulopathy was separated from thrombocytopenia using FIBTEM (maximum clot firmness 1.9 mm vs. 11.2 mm, P < 0.001). The run time of analysis to achieve diagnostic data was shorter applying a panel of TEM-reagents. A transfusion algorithm based on kaolin suggested platelets in case of dilutional coagulopathy, whereas an algorithm applying TEM-reagents suggested fibrinogen. Conclusion:Monoanalysis with kaolin was unable to distinguish coagulopathies caused by dilution from that of thrombocytopenia. Algorithms based on the use of kaolin may lead to unnecessary transfusion with platelets, whereas the application of TEM-reagents may result in goal-directed fibrinogen substitution.

Tranexamic acid combined with recombinant factor VIII increases clot resistance to accelerated fibrinolysis in severe hemophilia A

Background: Most patients with severe hemophilia A suffer from a profoundly compromised hemostatic response. In addition to both the delayed and slow development of a clot, previous studies have documented that severe hemophilia A is also associated with reduced clot stability.Objectives: We examined whether the clot stability in hemophiliacs could be improved by treatment with tranexamic acid (TXA) in combination with recombinant factor VIII (rFVIII).Patients/methods: Baseline blood samples were obtained from eight males with severe hemophilia A. Thereafter, a bolus injection of rFVIII was administered to increase the functional level of FVIII to ∼50%. After 10 min, blood was collected followed by an intravenous injection of TXA. A third blood sample was obtained after a further 10 min. Whole blood clotting profiles were determined by thrombelastography using minimal tissue factor activation. Clot formation was assessed by both clot initiation and clot propagation. At clot termination, the maximum clot firmness and area under the elasticity curve were used to illustrate clot stability. Tissue‐plasminogen activator was included in those experiments designed to assess clot stability. Results: As expected, rFVIII increased clot formation, whereas TXA had no effect upon this parameter. Assays including tissue‐plasminogen activator revealed that rFVIII increased the maximum clot firmness 3‐fold; whereas the presence of TXA induced an additional 4‐fold increase. The area under the elasticity curve increased 5‐fold after rFVIII and 24‐fold after addition of TXA. Conclusions: The study demonstrates that simultaneous treatment with TXA and rFVIII significantly improves the clot stability in patients with hemophilia A.

Management and monitoring of recombinant activated factor VII

&NA; Recombinant factor VIIa (rFVIIa) has recently been introduced as a new ‘bypassing’ agent to improve haemostasis in haemophilia patients with inhibitors to factor (F) VIII or FIX. In noninhibitor patients, levels of circulating FVIII or FIX can be used to assess the quality of substitution therapy. In contrast, laboratory monitoring of haemostatic efficacy in patients treated with rFVIIa has proved more complex. Evaluation of patient samples saved during rFVIIa treatment have revealed some correlation between FVII:C levels and improved haemostasis, while whole blood elasticity, as determined by thromboelastography, has been shown to improve following rFVIIa treatment. The investigation aimed to study the efficacy of rFVIIa in activating FX:C and in shortening the whole blood clotting time (WBCT) using the newly‐developed roTEG coagulation instrument. Results indicated a substantial and apparently dose‐dependent activation of FX:C by rFVIIa. In addition, the presence of FIX appeared to influence FX:C activation. In‐vitro and ex‐vivo roTEG thromboelastograph measurements showed that FVIIa shortened WBCT, although normalization did not occur. The results presented here are based on a small number of observations in a few patients and further studies should be planned to test the efficacy of these monitoring principles in clinical treatment practice with rFVIIa.

Thrombin generation and coagulation factor activities: evaluation and comparison with the international normalized ratio.

Oral anticoagulation therapy is monitored effectively by the international normalized ratio (INR). However, INR is perhaps not optimal in predicting risk of complications. Other more sensitive methods have been suggested. The aims of this study were to estimate the variability and association of calibrated automated thrombin generation (CAT) and clotting activity of coagulation factors II, VII, IX and X and to compare these parameters with the INR measured in a central laboratory as well as using portable coagulometers. Twenty-four patients on stable oral anticoagulation therapy with coumarins were followed prospectively for 6 weeks. A mixed effect model was used for the statistical analyses. The CAT and activity of coagulation factors II, VII, IX and X showed no significant difference in variability over time. Approximately 50% of the total variability of the CAT and coagulation factor activities was displayed as variation in the INR. The remaining variability was displayed as ‘within patient variation’. The CAT and coagulation factor activities were significantly associated with the INR. In conclusion, neither CAT nor coagulation factor activities seem to provide additional information about the biological variation; however, larger clinical studies are needed to investigate the ability to predict complications in individual patients on oral anticoagulation therapy.

Can RoTEM ® analysis be applied for haemostatic monitoring in paediatric congenital heart surgery?

BACKGROUND Successful management of bleeding disorders after congenital heart surgery requires detection of specific coagulation disturbances. Whole-blood rotation thromboelastometry (RoTEM®) provides continuous qualitative haemostatic profiles, and the technique has shown promising results in adult cardiac surgery. SETTING To compare the performance of RoTEM® with that of conventional coagulation tests in children, we conducted a descriptive study in children undergoing congenital cardiac surgery. For that purpose, 60 children were enrolled and had blood samples taken before, immediately after, and 1 day after surgery. Conventional coagulation tests included: activated partial thromboplastin time, prothrombin time, fibrinogen, fibrin D-dimer, thrombin clotting time, factor XIII, and platelet count. RESULTS Post-surgical haemostatic impairment was present to some degree in all children, as seen by pronounced changes in activated partial thromboplastin time, prothrombin time, thrombin clotting time, and platelet count, as well as RoTEM® analysis. RoTEM® showed marked changes in clotting time - prolonged by 7-18% - clot formation time - prolonged by 46-71% - maximum clot firmness - reduced by 10-19%, and maximum velocity - reduced by 29-39%. Comparison of the two techniques showed that conventional coagulation tests and RoTEM® performed equally well with regard to negative predictive values for excessive post-operative drain production - more than 20 millilitres per kilogram per 24 hours after surgery - with an area under the curve of approximately 0.65. CONCLUSION RoTEM® can detect haemostatic impairments in children undergoing cardiac surgery and the method should be considered as a supplement in the perioperative care of the children where targeted transfusion therapy is necessary to avoid volume overload.

Fibrinogen concentrate and cryoprecipitate but not fresh frozen plasma correct low fibrinogen concentrations following in vitro haemodilution

INTRODUCTION Fibrinogen deficiency often develops during massive bleeding due to e.g. fluid resuscitation with colloid plasma expanders like hydroxyethyl starch. This study investigates the haemostatic effect of various sources of fibrinogen: fibrinogen concentrates (Haemocomplettan® (FC 1), CSL Behring and Clottagen® (FC 2), LFB Biomedicaments), fresh frozen plasma (FFP), and Cryoprecipitate (CP). METHODS Whole blood samples provided by healthy individuals (n=8) were diluted 1:1 with HES or 0.9% isotonic saline as control. Various sources of fibrinogen was added to the HES diluted samples in concentrations equivalent to giving a 70 kg male either no correction, 2 grams of FC 1 or FC 2, 4 grams of FC 1 or FC 2, 15 ml/kg of FFP, 2 packs of CP or 4 packs of CP. Haemostatic effect was assessed by thromboelastometry initiated by tissue factor, with maximum clot firmness (MCF) as the primary endpoint. In addition thrombin generation was assessed for each intervention RESULTS HES dilution reduced MCF significantly more than isotonic saline. High dose FC 1, 2 and CP corrected the MCF so that it did not differ significantly from the isotonic saline dilution group. Thrombin generation following isotonic saline and HES dilution was comparable and not decreased compared to whole blood. Fibrinogen concentrates supplementation did not increase thrombin generation whereas CP and FFP both increased thrombin generation CONCLUSION Fibrinogen concentrates investigated dose dependently and equally corrected the MCF and caused no increase in thrombin generation. Cryoprecipitate also corrected the MCF, but also increased thrombin generation. FFP failed to improve MCF, but increased thrombin generation.

Ex-vivo response to blood products and haemostatic agents after paediatric cardiac surgery.

Bleeding complications after cardiac surgery are of particular importance in children because they are more prone to volume overload. To optimize haemostatic intervention, the coagulopathy has to be characterized, and knowledge about the effect of blood products and haemostatic agents is needed. The aims of the present study were to investigate changes in coagulation profiles after paediatric cardiac surgery and the effect after ex-vivo addition of blood products and haemostatic agents. Coagulation profiles were evaluated by thromboelastometry (ROTEM) in 54 children before and immediately after cardiac surgery. The haemostatic potential of various factor concentrates (fibrinogen concentrate, recombinant factor VIIa and factor XIII), fresh frozen plasma (FFP), pooled platelets and tranexamic acid was investigated. After surgery, the coagulation profiles revealed significantly prolonged clotting time (P = 0.008), and reduced clot propagation (P < 0.001) as well as reduced whole blood clot stability (P < 0.001). Ex-vivo addition of pooled platelets fully reversed the postoperative coagulopathy; this was also seen after addition of recombinant factor VIIa although less pronounced. Finally, addition of fibrinogen concentrate, FFP or tranexamic acid improved clot stability significantly. Whole blood coagulation was significantly impaired after cardiac surgery in children. Ex-vivo studies showed a total reversal of the coagulopathy after addition of pooled platelets and significantly improved clot stability after addition of fibrinogen concentrate, FFP and tranexamic acid, respectively.

Evaluation of coagulation kinetics using thromboelastometry—methodologic influence of activator and test medium

Renewed interest has arisen in the use of thromboelastography/thromboelastometry in evaluating coagulation kinetics. The test medium, type of activator, and its concentration may influence the interpretation of coagulation kinetics. This study aimed to investigate methodologic influences of activator and test medium on thromboelastometric parameters of coagulation kinetics. Dynamic clot formation was evaluated by thromboelastometry using whole blood (WB), platelet-rich plasma, or platelet-poor plasma employing different concentrations of extrinsic (tissue factor) and contact activator (synthasil) and with variable concentrations of phospholipids. Plasma samples displayed prolonged clot initiation and enhanced clot propagation compared with WB. Clot firmness was markedly reduced in platelet-poor plasma as compared with platelet-rich plasma and WB. Increasing concentration of activator shortened the clot initiation and increased the velocity of clot propagation, whereas terminal clot firmness remained unaffected. Platelets accelerated clot propagation and raised clot firmness. Phospholipids shortened the time of clot initiation and increased velocity of propagation, while clot firmness remained unchanged. Our results demonstrate that evaluation of coagulation kinetics using thromboelastometry varies according to the composition of the test medium, type, and concentration of activator, as well as the presence and concentration of phospholipids in the test reagent.

Platelet activation and aggregation: the importance of thrombin activity—A laboratory model

This study introduces a new laboratory model of whole blood platelet aggregation stimulated by endogenously generated thrombin, and explores this aspect in haemophilia A in which impaired thrombin generation is a major hallmark. The method was established to measure platelet aggregation initiated by tissue factor evaluated by means of impedance aggregometry. Citrated whole blood from healthy volunteers and haemophilia A patients with the addition of inhibitors of the contact pathway and fibrin polymerization was evaluated. In healthy persons, a second wave of platelet aggregation was found to coincide with the thrombin burst and to be abolished by thrombin inhibitors. In this system, platelet aggregation in severe haemophilia A (n = 10) was found to be significantly decreased as compared with healthy individuals (912 ± 294 vs. 1917 ± 793 AU × min, P = 0.003), most probably due to the weak level of thrombin generation. For the first time, analysis of platelet aggregation as induced by endogenously generated thrombin was demonstrated. The new method makes it possible to explore the influence of the coagulation system on platelet function. In contrast to the general understanding, the data suggest that the impaired thrombin generation in haemophilia may affect platelet activation. Future studies will address whether our results may contribute to understanding differences in bleeding phenotypes and response to haemostatic substitution observed among patients.