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INTRODUCTION A dedicated emicizumab assay based on the modified one-stage factor VIII (FVIII) assay (mOSA) is mainly available in haemophilia treatment centres (HTC). A method to estimate emicizumab plasma levels based on a widely available assay would be desirable, especially for emergency situations. AIM A method for emicizumab plasma level approximation (ELA) using a routine FVIII activity measurement with standard one-stage assay (sOSA) was developed and evaluated. METHOD Within this pilot study, 59 samples from patients with severe haemophilia A with (n = 8) and without (n = 8) inhibitors under emicizumab treatment were analysed using sOSA following a manual 1:8 sample pre-test dilution with saline. The sOSA was determined in two different laboratories, using two different analyser platforms each. RESULTS The results demonstrated an excellent correlation of approximated emicizumab plasma levels (ELA) with the emicizumab plasma concentration determined with mOSA (r > .9; p

BACKGROUND: Platelets play a pivotal role in coagulation, inflammation and wound healing. Suitable animal models that have the potential to mimic human platelet function are limited. The objective of the current study was to compare platelet aggregation response in the whole blood of baboons and humans using impedance aggregometry.METHODS: Blood was drawn from 24 anesthetised male baboons and 25 healthy volunteers. The platelet aggregation response was determined by impedance aggregometry (Multiplate®). Platelets in the hirudinised whole blood samples were stimulated with four different activators: adenosine diphosphate (ADP), collagen (COL), thrombin receptor activating peptide-6 (TR1AP), and activation of PAR-4 thrombin receptor subtype (TR4AP) at standard concentrations. Higher than standard concentrations were tested in a subgroup of the animals.RESULTS: The cell counts showed no differences between baboons and humans. The platelet aggregation response was significantly lower in baboons compared to humans when stimulated with the platelet agonists ADP (pCONCLUSION: The current study revealed that testing the platelet function in baboon blood by impedance aggregometry is feasible with ADP, COL and TR4AP, but not with TR1AP. Compared to humans, the aggregation response is lower in baboons. Considering the limitations in accordance to these results, baboons might represent a potential species for further platelet research.

Platelets are playing a crucial role in acute cardiovascular events. We investigated if physical stress activates platelets and whether this activation can be inhibited by a polyphenol-enriched diet. Blood samples were taken from a total of 103 athletes three weeks before, one day before, immediately as well as 24 hours and 72 hours after a marathon run. Participants were randomized, double-blinded and divided into two groups. One group received a polyphenol-rich beverage the other the same beverage without polyphenols. Besides analysis of platelet counts and impedance-aggregometric-measurement of platelet activity, soluble P-selectin and Endothelin-A measurements were performed. In the control group, runners showed a 2.2-fold increased platelet aggregation directly after completing a marathon and within the following three days when compared with baseline values (p

Adenosine 5′-diphosphate (ADP) inducible aggregation is used to assess platelet response to thienopyridines. Thrombin receptor-activating peptide-6 (TRAP-6) inducible aggregation may serve as a positive control because it acts via the thrombin receptor protease-activating receptor-1, which is not blocked by thienopyridines. We therefore investigated if TRAP-6 is suitable as a positive control when assessing residual platelet reactivity to ADP. Platelet response to clopidogrel was assessed in 200 patients on dual antiplatelet therapy using ADP inducible platelet aggregation by light transmission aggregometry (LTA), multiple electrode aggregometry (MEA), and the shear-dependent Impact-R. Test specificities were monitored by TRAP-6 inducible platelet aggregation. The aggregation-independent vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay served for comparisons. ADP inducible aggregation was correlated to that by TRAP-6 (r = 0.33 to 0.72; p0.001 for all assays). A linear correlation was seen within MEA (r = 0.72). LTA TRAP-6 correlated weakly with the VASP assay (r = 0.19; p = 0.01), while there were no correlations of TRAP-6 responses by MEA or the Impact-R with the VASP assay (r = 0.03 and#x2212;0.09; p0.05). In all three assays, differences between ADP and TRAP-6 inducible aggregation varied considerably. Within MEA, TRAP-6 inducible aggregation was almost always stronger than ADP inducible aggregation, while within LTA and the Impact-R, weak responses to ADP were associated with both, weak and strong responses to TRAP-6. In conclusion, the application of TRAP-6 as a positive control for platelet reactivity has major limitations and results need to be cautiously interpreted on an individual basis.

Our aim was to assess the change in platelet activity along the menstrual cycle. We conducted a prospective observational study. The study group included 16 healthy women with regular menstrual cycles, which were compared to a control group of 14 healthy males. Exclusion criteria were age18 years or45 years, use of oral contraceptives or any other forms of hormonal therapy and medical disorders or medications that might affect platelet aggregation. Blood samples were taken from each of the women at four different phases of the menstrual cycle: day 1 +/- 1, day 7 +/- 1, day 14 +/- 1, and day 21 +/- 1. A single blood sample was taken from the males. Platelet aggregation was assessed in whole blood samples using the Multiplate analyzer with three different agonists (ADP, arachidonic acid (AA), and thrombin-receptor activating peptide (TRAP)). Platelet aggregation for each of the women at each of the phases of the menstrual cycle was expressed as the percentage change from the day 1 +/- 1 value. A total of 390 aggregation assays were performed. The mean aggregation activity was significantly higher in females compared with males, irrespective of the agonist used. For the TRAP and the ADP agonists, the relative platelet activity decreased along the menstrual cycle from day 1 towards day 21 and from day 7 towards day 21, respectively, although differences reached statistical significance only for day 21 (-12.4% +/- 3.2%, P0.05 for TRAP, and -9.5% +/- 3.9%, P0.05 for ADP). When using AA to induce platelet aggregation, the relative platelet activity was highest around the time of ovulation (11.0% +/- 4.7%) and was significantly lower on day 21 (-8.5% +/- 6.7%, P0.05). In conclusion, platelet aggregation activity is higher in females compared with males. The association between the phase of the menstrual cycle and platelet activity appears to vary with the type of agonist, but platelet aggregation is consistently lowest in the mid-luteal phase irrespective of the agonist used.

Of superior importance for the novel concepts of activation of blood coagulation are the interactions of blood cells, the plasmatic coagulation factors and the dynamics of thrombin formation. A novel model of overlapping phases of activation and intensification of hemostatic processes has now replaced the outdated ”classical„ model of activation of coagulation by the coagulation cascade and its extrinsic and intrinsic system. Parallel to the growing understanding of the system of coagulation, novel routines of laboratory chemical analysis - which are often conducted with full blood - have been developed. Furthermore, also the classical global tests PT (prothrombin time) and aPTT (activated partial thromboplastin time) are being increasingly performed with full blood, i.e. close to the patient. The following contribution presents new methods for measuring hemostasis that are specifically employed in the clinical control of hemostastically relevant therapies.

Background: Platelet dysfunction contributes to the pathophysiology of bleeding complications during and after cardiac surgery. In most surgical institutions, no peri-operative point-of-care monitoring of platelet function is used. We evaluated the usefulness of the Multiplate® platelet function analyser based on impedance aggregometry for identifying groups of patients at a high risk of transfusion of platelet concentrates (PC). Methods: Platelet function parameters were determined in 60 patients before and after routine cardiac surgery. Impedance aggregometry measurements were performed on Multiplate® using ADP (ADPtest), collagen (COLtest) and thrombin receptor activating peptide (TRAPtest) as platelet activators. The correlations between the aggregometry results and the transfusion of PC were calculated. The results of the aggregation tests were also divided into tertiles and the differences in PC transfusion between the low and the high tertile were assessed. Results: Low aggregometry delimited groups of patients with significantly higher PC transfusion. In the receiver operating characteristic curve, low pre-operative aggregation in the ADPtest identified patients with high total transfusion of PC (area under the curve 0.74, P=0.001), while the ADPtest performed at the end of the operation identified patients with high PC transfusion on the intensive care unit (ICU) (area under the curve 0.76, P=0.002). Conclusions: Near-patient platelet aggregation may allow the identification of patients with enhanced risk of PC transfusion, both pre-operatively and upon arrival on the ICU.

SummaryThis article provides an overview on current commercially available methods to determine primary haemostasis as a target of drug-mediated anticoagulation. It focuses on whole blood methods only, and references the currently major achievements that have been reported with each method in respect to its clinical use. Advantages and disadvantages of the various methods are presented, based on considerations of platelet physiology, and on feasibility of the procedures.

Platelet dysfunction due to antiplatelet therapy contributes to perioperative bleeding. Several trials investigating the influence of aspirin intake within the 5 days before surgery reported that transfusion requirements were either increased or not significantly affected by aspirin intake. Our objective was to compare the assessment of aspirin intake by patient self-reporting and by measurement of platelet function with regard to transfusion requirements.In a prospective trial, a standardized questionnaire was used in 100 patients for aspirin intake within the 5 days immediately before coronary artery bypass grafting. Whole blood platelet aggregation triggered by arachidonic acid was investigated using the Multiplate platelet function analyzer.Eleven of 23 patients with aspirin intake within the 5 days before the intervention showed an abnormal aggregation response. Nine of 77 patients who reported no aspirin intake before surgery had an abnormal aggregation response. There were no significant differences in chest tube drainage and red blood cell transfusion over the first 24 h postoperatively between patients with and without reported aspirin intake. There was no significant difference in chest tube drainage over the first 24 h postoperatively between patients showing normal or abnormal aggregation response. Patients with abnormal aggregation before intervention (51 U) received significantly more platelet transfusion than patients with normal aggregation (1.1 U compared to 0.3 U, P = 0.001).Our results suggest that arachidonic acid-induced aggregation in whole blood may be a better predictor of platelet-related coagulopathy and platelet transfusion than the assessment of aspirin intake by patient self-reporting.

The prothrombinase-induced clotting time assay (PiCT, Pentapharm, Basel, Switzerland) is a clotting assay sensitive to factor Xa and factor IIa inhibitors. It is based on the addition of factor Xa and snake venom RVV-V (Russell viper venom factor V activator) specifically activating factor V and phospholipids to platelet-poor plasma. Following an incubation time, the mixture is recalcified and the clotting time is determined. An almost linear dose-response and high sensitivity of the assay for unfractionated heparin (UFH), low-molecular-weight heparins (LMWHs), r-hirudin, and argatroban was found. Fondaparinux showed a nonlinear dose-response. By using ex vivo samples, the following Pearson correlation coefficients were found: r = 0.85 between amidolytic anti-Xa and PiCT for 120 LMWH and 24 control samples; r = 0.86 between amidolytic anti-Xa activity and PiCT for 68 UFH and 24 control samples; and r = 0.94 between ECT and PiCT for 38 hirudin samples. Thus, PiCT is a promising assay for the monitoring of anticoagulants inhibiting factor Xa and/or factor IIa.

BACKGROUND: Recombinant activated factor VII (rFVIIa) is increasingly used for treating refractory bleeding after cardiac surgery. However, hemostasis also depends on coagulation factors, including fibrinogen, which stabilizes platelet plugs at sites of vascular injury. We compared the hemostatic effects of rFVIIa, fibrinogen, or their combination. METHODS: Blood samples were obtained from 12 volunteers and from 7 patients after cardiopulmonary bypass (CPB). The in vitro effects of rFVIIa (1.5 μg/mL), fibrinogen (100 mg/dL), and the combination were evaluated under simulated coagulopathy in volunteer plasma using heparin (0.1 U/mL) or tissue plasminogen activator (0.1 μg/mL). Hemostatic interventions were compared using thromboelastometry, which measures clotting time (CT, s), angle of thrombus formation, and maximal clot firmness (MCF, mm). The Thrombinoscope™ was used to quantitate thrombin generation after addition of fibrinogen and/or rFVIIa. RESULTS: In heparinized volunteer plasma, rFVIIa shortened CT (1st and 3rd quartiles) from 663 (522–736) to 435 (397–531) s, but it did not affect MCF. Fibrinogen increased MCF from 26.0 (24.4–26.7) to 30.5 (26.3–31.5) mm without affecting CT. The combination of rFVIIa and fibrinogen in heparinized samples was most effective in improving CT to 359 (324–522) s and MCF to 29 (27.8–31.0) mm. In tissue plasminogen activator-treated volunteer plasma, fibrinolysis increased by more than 45% by the addition of rFVIIa. After CPB, both CT and MCF were most improved with coadministration of rFVIIa and fibrinogen. Thrombinoscope evaluation demonstrated that rFVIIa decreased the lag time and increased peak thrombin generation, whereas fibrinogen had no effect. CONCLUSION: The onset of fibrin formation and thrombin generation were shortened after rFVIIa addition, but fibrin clot strength was only increased after fibrinogen supplementation. In vitro clot formation was most improved by using both rFVIIa and fibrinogen in whole blood after CPB.

ZusammenfassungIn dieser Studie wurden zwei Methoden evaluiert und verglichen, welche die Wirkung von Azetylsalizylsäure (ASS) auf die Thrombozytenaggregation im Vollblut erfassen. Die In-vitro-Blutungszeit wurde im PFA-100 (Dade Behring, Marburg) mit der Kollagen/Adrenalin-Messzelle im Zitratblut bestimmt. Die Vollblutaggregation wurde am Multiplate- System (Dynabyte medical, München) mit Hirudinblut (25 μg/ml) bestimmt. Zur Aggregation wurden die Induktoren Arachidonsäure (ASPItest), Kollagen (COLtest) sowie TRAP-6 (thrombin receptor activating peptide, TRAPtest) eingesetzt. Nach Aufklärung und Einwilligung wurden zur Analyse der In-vitro-Blutungszeit sowie der Vollblutaggregometrie Zitratblut und Hirudinblut von 76 Patienten mit koronarer Herzkrankheit, die unter langfristiger Azetylsalizylsäure- Behandlung standen (ASS-Patienten), entnommen. Zusätzlich wurde von 57 gesunden Blutspendern Hirudinblut für die Vollblutaggregometrie entnommen.Im PFA-100 betrugen die Verschlusszeiten der ASS-Patienten 273 ± 49 s. Basierend auf dem Grenzwert (cut-off) von 170 s wurde bei 5 von 76 Patienten kein Ansprechen des Tests auf die ASS-Therapie nachgewiesen (Aspirin-Non-Response). Die Vollblutaggregometrie war bei den ASS-Patienten im Vergleich (vs. AUC-Werte) zu den Blutspendern in TRAPtest vergleichbar, hingegen im COLtest und ASPItest signifikant vermindert (p Die in der Studie gefundenen hohen Ansprechraten des PFA-100 sowie ASPItest auf die Azetylsalizylsäuretherapie kann durch die erwartete gute Compliance im untersuchten Kollektiv erklärt werden. In den verschiedenen Tests wurden unterschiedliche Patienten als Non-Responder zugeordnet. Dies verdeutlicht die Bedeutung der Testbedingungen für die Diagnose einer Azetylsalizylsäure-Resistenz oder Aspirin- non-Response.

Severe bleeding often induces coagulopathy via loss, consumption, and dilution of clotting factors and platelets. The aims of our in vitro study were to characterize the influence of progressive hemodilution with either NaCl 0.9% or hydroxyethyl starch (HES) 6% on blood clot formation and to analyze the effect of substitution of fibrinogen and platelets on dilutional coagulopathy. Whole blood samples drawn from 8 volunteers were diluted from 20% to 80% of the sample volume with both diluents separately. Clot formation was measured by thrombelastography. At a 60% dilution, either fibrinogen and/or platelets were added to the samples. Clot firmness became critical after 40% dilution with HES 6% but not until 60% dilution with NaCl 0.9%. When platelet function was blocked, fibrin polymerization was severely impaired after 20% dilution with HES 6%, whereas such an effect was only seen after 80% dilution with NaCl 0.9%. The addition of fibrinogen reconstituted the clot firmness in the presence of NaCl 0.9%, but this had only a minor effect after dilution with HES 6%. Platelets alone or in addition were not able to improve clot firmness to a clinically relevant extent. Dilutional coagulopathy induced by crystalloids can, in vitro, be effectively reversed by supplementation of fibrinogen. In contrast, HES molecules interfere with fibrin polymerization and, thus, administration of fibrinogen after dilution with HES 6% failed to significantly improve clot firmness.

A new prothrombin-based activated protein C resistance (APC-R) test is described. In this method, the patient sample is prediluted in a plasma depleted of factor V (FV). A reagent containing APC and a specific activator of FV is added. After an incubation period, clotting is initiated by the addition of the FV-dependent prothrombin activator Noscarin. We analyzed 703 samples from patients undergoing thrombophilia screening. By using a predefined cutoff ratio of 2.5, 100% sensitivity and specificity for the detection of a factor V Leiden (FVL) mutation was found. With a cutoff ratio of 1.2, a complete but narrow distinction of FVL heterozygous (n = 192) and FVL homozygous samples (n = 27) was determined. No interference by the international normalized ratio, activated partial thromboplastin time (aPTT), protein S activity, fibrinogen and factor VIII (FVIII) levels, or lupus anticoagulant ratio was detected. The new prothrombin-based APC-R assay provides improved distinction of FV wild-type and FVL carriers compared with the aPTT-based method. By the use of an FV-dependent prothrombin activator, the assay is not influenced by FVIII concentration or lupus anticoagulants.

Zusammenfassung: Eine Reihe von hamostaseologischen Point of Care (POC)-Verfahren hat vor allem in den letzten Jahren ihren Einsatz in der Klinik und im Selbstmanagement des Patienten gefunden. Das Spektrum der verfugbaren Methoden erstreckt sich von einfachen Instrumenten zur Erfassung der plasmatischen Gerinnung (ACT, CoaguCheck, Rapidpoint Coag), uber Thrombozytenfunktionstests (PFA-100, RPFA) bis hin zu den komplexeren viskoelastischen Analyseverfahren (TEG, ROTEG, Sonoclot). Der entscheidende Vorteil der POC-Analyse ist die schnelle Verfugbarkeit der Ergebnisse mit der Moglichkeit der zielgerichteten therapeutischen Intervention. Nutzen und Risiken der POC-Verfahren werden entscheidend von der Dringlichkeit der Analyse, der Response-Zeit des Labors, den verfugbaren personellen und logistischen Ressourcen sowie von dem zu erwartenden Spektrum der Gerinnungsveranderungen gepragt. Bei der Steuerung der Antikoagulation mittels Heparin oder Hirudin ist die POC-Analyse haufig sinnvoll und hat sich hier entsprechend weit durchgesetzt. Die Steuerung des Hamostasemanagements bei akuten Blutungen, sowie operativen Eingriffen mit hohem Transfusionsbedarf mittels der POC-Analytik ist vor allem im angloamerikanischen Raum weit verbreitet. Ein haufig vernachlassigter Aspekt bei der POC-Analyse ist die Qualitatssicherung. Da Vollblut nicht uber langere Zeit stabil ist und auch nicht lyophilierbar ist, werden Kontrollmaterialien auf der Basis von Plasma, artifizielle Flussigkeiten sowie elektronische und on-board Verfahren eingesetzt. Die Einfuhrung und der Betrieb von POC-Methoden stellt die klinisch tatigen Kollegen vor neue Herausforderungen, wobei die schnelle Diagnostik mit der Moglichkeit der zielgerichteten Therapie den Einsatz dieser Methoden sinnvoll und kosteneffektiv gestalten kann. Summary: Several Point-of-Care (POC) methods for the analysis of the hemostatic system have been introduced into clinical applications during the last years. Available methods include simple instruments for the assessment of plasmatic coagulation (ACT, CoaguCheck, Rapidpoint Coag), specific platelet function assays (PFA-100, RPFA), and the more complex visco-elastic assays (TEG, ROTEG, Sonoclot). The main advantage of POC analysis is the short turn-around time which allows a targeted hemostasis management. The benefit and the risks of POC analysis depend on the urgency of the analysis, the turn-around time of the laboratory analysis, the availability of motivated staff at the point of care and the expected hemostatic alterations. The use of POC analysis for the monitoring of heparin and hirudin is often justified and is widely applied. The monitoring-directed management of acute bleeding complications using POC analysis is in particular popular in the english-speaking countries, while it is less frequently applied in central Europe. An underestimated aspect of POC analysis is the importance of established quality management procedures. As whole blood is not stable for longer periods and cannot be lyophilizied without large alterations, control materials based on plasma, artificial fluids, and electronic on-board methods are applied. In conclusion the introduction and the application of POC methods provides new tasks and challenges. The fast analysis of the hemostasic system and the ability for targeted therapeutic approaches can make this approach useful and economically effective.

Background: Hydroxyethyl starches (HES) are known to interfere with blood coagulation according to molecular weight, the degree of substitution and the C2/C6 ratio. A recently developed low molecular hydroxyethyl starch (HES 130/0.4) was designed to reduce the blood compromising potency. Methods: In this study, effects of a 30% in vitro haemodilution with the new HES preparation (HES 130/0.4) in comparison to HES 200/0.5, HES 450/0.7 and sodium chloride solution were investigated using intrinsic and extrinsic activated thrombelastography (TEG) and plasmatic coagulation tests. Results: Whereas plasmatic tests revealed no prolongation of coagulation by HES in comparison to sodium chloride, the TEG variables clotting time, clot formation time and maximal clot firmness showed a significant (P

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.

Background The question of whether novel instruments such as multiple electrode aggregometry (MEA) can be used for measurement of the effects of nitric oxide (NO) on platelets (PLTs) has not been examined. Methods Therefore, we compared the effects of NO concentrations (1, 10, and 100 microM) on the PLT aggregation response to ADP, arachidonic acid (AA), collagen, ristocetin, and thrombin receptor-activating peptide 6 (TRAP6) using light transmission aggregometry (LTA) and multiple electrode aggregometry (MEA) and examined the effects of NO using the platelet function analyzer (PFA)-100. Results The response of PLTs to ADP and AA was strongly inhibited by all NO concentrations in LTA and MEA. The inhibition of the responses to ristocetin and collagen was detectable in MEA at lower NO concentrations than in LTA. However, the typically increasing lag phase between collagen addition and the aggregation response in the presence of NO was more obvious in LTA. TRAP caused a reproducible early response in the presence of NO in LTA which was followed by rapid PLT disaggregation, whereas even 100 microM NO did not inhibit the response to TRAP in MEA. Finally, NO prolonged the in-vitro bleeding time remarkably more in the PFA-100 collagen-epinephrin cartridge than in the collagen-ADP cartridge. Conclusions Whole blood versus PLT rich plasma, citrate versus hirudin, and high versus low shear influenced the effects of NO. This shows that a careful selection of models and potentially a combination of different methods is appropriate for a differentiated evaluation of pharmacological or physiological mechanisms of NO-donors or of NO-inhibitors.

Patients undergoing coronary artery bypass grafting (CABG) are at risk of postoperative bleeding because of decreased platelet function and cardiopulmonary bypass (CPB)-induced haemostatic impairment. Fibrinogen concentration decreases by 34-42% of the preoperative level by the end of CPB. An inverse relationship between perioperative plasma fibrinogen levels and postoperative bleeding has been reported in CABG patients. Administration of fibrinogen concentrate after weaning from CPB in patients with diffuse microvascular bleeding may help promote haemostasis. We compared patient outcomes following fibrinogen concentrate administration or transfusion of allogeneics in CABG patients with decreased platelet function.Thirty-eight patients with decreased preoperative platelet function in Multiplate aggregometry were included. Patients with bleeding after CPB received either fibrinogen concentrate (guided by the measurement of fibrin clot quality using the FIBTEM thromboelastometric test) or allogeneics.Twenty-nine of 38 patients received haemostatic therapy (bleeding + fibrinogen group, n = 10; bleeding + allogeneics group, n = 19). Total transfusion (median (interquartile range)) was significantly lower in the bleeding + fibrinogen group (0 (0, 3.8) units), compared with the bleeding + allogeneics group (6 (5, 8) units, p = 0.0073). Bolus administration of fibrinogen concentrate increased FIBTEM maximum clot firmness from 10.5 (9.3, 11) mm after CPB to 20.5 (20, 21.8) mm at the end of surgery. Postoperative outcomes were similar in both groups. No treatment-related complications were observed after fibrinogen concentrate.In CABG patients with bleeding after CPB, FIBTEM-guided administration of fibrinogen concentrate resulted in overall decreased transfusion, compared with haemostatic therapy with allogeneics. Fibrinogen concentrate administration increased the fibrin clot quality and helped achieve haemostasis.

Platelet dysfunction contributes to the pathophysiology of bleeding complications during and after cardiac surgery. In most surgical institutions, no peri-operative point-of-care monitoring of platelet function is used. We evaluated the usefulness of the Multiplate platelet function analyser based on impedance aggregometry for identifying groups of patients at a high risk of transfusion of platelet concentrates (PC).Platelet function parameters were determined in 60 patients before and after routine cardiac surgery. Impedance aggregometry measurements were performed on Multiplate using ADP (ADPtest), collagen (COLtest) and thrombin receptor activating peptide (TRAPtest) as platelet activators. The correlations between the aggregometry results and the transfusion of PC were calculated. The results of the aggregation tests were also divided into tertiles and the differences in PC transfusion between the low and the high tertile were assessed.Low aggregometry delimited groups of patients with significantly higher PC transfusion. In the receiver operating characteristic curve, low pre-operative aggregation in the ADPtest identified patients with high total transfusion of PC (area under the curve 0.74, P=0.001), while the ADPtest performed at the end of the operation identified patients with high PC transfusion on the intensive care unit (ICU) (area under the curve 0.76, P=0.002).Near-patient platelet aggregation may allow the identification of patients with enhanced risk of PC transfusion, both pre-operatively and upon arrival on the ICU.

Thromb Haemost 2009; 101: 12–13 The combination of aspirin with clopidogrel is currently the mainstay of antiplatelet therapy for patients with acute coronary syndromes (ACS) (1). While aspirin inhibits platelet thromboxane A2 production and platelet activation, clopidogrel inhibits adenosine diphosphate (ADP)-induced platelet activation by blocking the P2Y12 platelet receptor. When added to aspirin therapy in patients with ACS, clopidogrel significantly reduces the risk of recurrent ischaemic events (1). Clopidogrel s onset of action is delayed and variable between individuals. A therapeutic level of inhibition is reached 4–6 hours (h) after a 300 mg loading dose, and 2 h after a 600 mg loading dose. Laboratory analysis reveals that a substantial proportion of patients do not show adequate inhibition of ADP-induced aggregation. Low responsiveness to clopidogrel has been shown to be significantly associated with adverse outcomes after percutaneous coronary intervention (PCI) (2). Prasugrel is a novel thienopyridine pro-drug with a greater rate, magnitude, and consistency of platelet ADP inhibition, as compared to clopidogrel. The therapeutic level of the inhibition of platelet aggregation is reached within 1 h after a 60 mg loading dose (3). TRITON-TIMI 38 (4) validated the hypothesis that more intensive antiplatelet blockade via the ADP receptor decreases the incidence of ischaemic events. Patients (13,608) with acute coronary syndromes scheduled for PCI and receiving aspirin were randomly assigned to receive either prasugrel or clopidogrel. After a median duration of 14.5 months, the primary efficacy outcome of cardiovascular death, non-fatal myocardial infarction, or non-fatal stroke occurred in 12.1% of patients taking clopidogrel and in 9.9% of those taking prasugrel. Stent thrombosis was also reduced in the prasugrel group (2.4% clopidogrel vs. 1.1% prasugrel). However, major bleeding was increased (1.7% vs. 2.5%), as was fatal bleeding (0.1% vs. 0.4%) in the prasugrel group. Overall mortality did not differ significantly between treatment groups. In their viewpoint article, Serebruany et al. (5) discuss different aspects of the clinical development of prasugrel and, in particular, the pivotal trial TRITON-TIMI-38. Although their interpretations and evaluations are definitely debatable, this article highlights several points which are of outstanding interest for clinical and research approaches to platelet function inhibition: – Standardization: Serebruany et al. (5) highlight two aspects of standardization. First, the standardization of definitions of endpoints for clinical trials; in order to be able to compare event rates in different studies, a standardized definition of endpoints is desirable. Second, standardization of monitoring techniques for the assessment of anti-platelet therapy is crucial. The authors comment on discrepancies that occurred in the early phase of clinical development of prasugrel, which may be attributed to the use of different equipment for aggregation analysis in participating centers. Today, there is still a wide variation in the literature and clinical practice with respect to instruments, reagents, sample preparation, sample anticoagulants, as well as the analysis algorithms and clinical cut-offs applied for anti-platelet drug monitoring. – Patient compliance: An intensified anti-platelet treatment is usually equalized with a decreased rate of low patient responsiveness to anti-platelet treatment. Serebruany et al. (5) remind us that in a real-life scenario, patient compliance is crucial. Intensifying anti-platelet therapy might generate an increased rate of annoying minor bleeding complications during everyday activities, such as shaving or brushing teeth, which may again lead to patient non-compliance. For the physician, this includes the comforting fact that even with more modern and intensified anti-platelet regimens, the role of personal communication with the patient is still important for the appropriate adherence of the patient to his life-saving treatment. – Focus on safety: With the growing efficacy of antithrombotic treatments in ACS and in other areas such as prophylaxis of deep venous thrombosis, there is an increased focus on drug safety. Melagatran and ticlopidine are examples of antithrombotic drugs that were either withdrawn from the market, or experienced a sharp drop in use due to safety concerns. Referring to prasugrel, Bhatt (6) states that in a “realworld” setting, such as an elderly patient with multiple coexisting conditions, the risk of major bleeding and even fatal bleeding may increase to an even greater degree than was seen in TRITON–TIMI 38. This expectation is also expressed by Serebruany et al. in their viewpoint article (5). – Transformation of clinical trials into routine patient management: Clinical trials have inclusion and exclusion © 2009 Schattauer GmbH, Stuttgart

Cristina Solomon1, Michael Winterhalter1, Isabel Gilde1, Ludwig Hoy2, Andreas Calatzis3, Niels Rahe-Meyer11Department of Anesthesiology, Hannover Medical School, Hannover, Germany; 2Institute for Biometry, Hannover Medical School, Hannover, Germany; 3Department Hemostasis Transfusion Medicine, University Hospital Munich, Munich, GermanyBackground: The standard method of assessment of platelet function is represented by light transmission aggregometry (LTA), performed in citrated platelet-rich plasma (PRP). With LTA, decrease and subsequent post-cardiopulmonary bypass (CPB) recovery of platelet function have been reported during cardiac surgery. Multiple electrode aggregometry (MEA) may be used as point-of-care method to monitor perioperative changes in platelet function. Since MEA assesses macroaggregation which is influenced by the plasmatic levels of unbound calcium, citrate may be inadequate as anticoagulant for MEA. We used citrate and heparin for MEA samples, to see with which anticoagulant the intraoperative decrease and postoperative recovery in platelet function previously described with other aggregometric methods in cardiac surgery may be observed with MEA.Methods: Blood was obtained from 60 patients undergoing routine cardiac surgery and the samples were collected in standard tubes containing unfractionated heparin (50 U/mL) or trisodium citrate (3.2%). The samples were obtained before CPB, at 30 minutes on CPB, end of CPB and on the first postoperative day. MEA was performed using the Multiplate® analyzer. Collagen (COLtest, 100 μg/mL) and TRAP-6 (thrombin receptor activating peptide, TRAPtest, 1mM/mL) were used as aggregation agonists.Results: Platelet aggregometric response decreased significantly during CPB. Platelet aggregation assessed using TRAP-6 as agonist on heparinized blood significantly correlated with the duration of CPB (r = −0.41, p = 0.001, 2-tailed Pearson test). The aggregometric analysis performed on the first postoperative day showed a significant recovery in platelet activity in the samples containing heparin (increase from 30 ± 22 U to 46 ± 27 U for the COLtest and from 70 ± 34 U to 95 ± 32 U for the TRAPtest, p

In this study two aspirin sensitive platelet function tests which are based on the analysis of whole blood were evaluated and correlated with each other. In vitro bleeding time was determined using the PFA-100 analyzer (Dade Behring, Marburg, Germany) using the collagen/epinephrine cartridge and citrated blood. Whole blood aggregometry was performed using the Multiplate analyzer (Dynabyte medical, Munich, Germany) using hirudin blood (25 mug/ml). Aggregatin was triggered using arachidonic acid (ASPItest), collagen (COLtest) or TRAP-6 (thrombin receptor activating peptide, TRAPtest). Following informed consent citrated blood and hirudin blood was drawn from 76 cardiovascular patients which were on long-term aspirin therapy (aspirin patients). In addition hirudin blood was drawn from 57 healthy blood donors for assessment of whole blood aggregometry. PFA-100 closure times of the aspirin patients were 273 +/- 49 s. Based on the cut-off of 170 s a non response to the aspirin therapy was detected in 5 of 76 patients. Whole blood aggregation was comparable in the aspirin patients vs the blood donors AUC values in the TRAP test, whereas in COLtest and ASPItest significantly reduced aggregations were detected (p0.05). Of the five patients that had a normal PFA-100 closure time only one had normal aggregation in ASPItest, and also only one had a normal aggregation in COLtest. The high rate of response to the aspirin therapy which was found in PFA-100 and ASPItest can be explained by the assumed high level of compliance of the cohort. In the applied tests different patients were stratified as aspirin-non-responders. This highlights the importance of the assay conditions for the diagnosis of an aspirin-non-response.

SummarySeveral methods are used to analyse platelet function in whole blood. A new device to measure whole blood platelet aggregation has been developed, called multiple electrode platelet aggregometry (MEA). Our aim was to evaluate MEA in comparison with the single platelet counting (SPC) method for the measurement of platelet aggregation and platelet inhibition by aspirin or apyrase in diluted whole blood. Platelet aggregation induced by different concentrations of ADP, collagen and TRAP-6 and platelet inhibition by apyrase or aspirin were determined in citrateor hirudin-anticoagulated blood by MEA and SPC. MEA indicated that spontaneous platelet aggregation was lower, and stimulated platelet aggregation was higher in hirudin- than citrate-anticoagulated blood. In hirudin-anticoagulated, but not citrate-anticoagulated blood, spontaneous platelet aggregation measured by MEA was inhibited by apyrase. For MEA compared with SPC the dose response-curves of agonist-induced platelet aggregation in citrate- and hirudin-blood showed similar EC50 values for TRAP, and higher EC50 values for ADP (non-significant) and collagen (p

In recent years point-of-care testing (POCT) has seen wider applications in the clinical management of surgical and critically ill patients. The available methods for haemostasis analysis include simple-to-handle tests for the assessment of plasmatic coagulation, platelet function tests and the more complex visco-elastic assays. The main advantage of POCT is the fast availability of the results allowing a targeted management of haemostasis disorders. The benefits and risks of POCT depend on the urgency of the analysis, the turn-around time of the laboratory tests, the availability of motivated staff at the point-of-care and the expected haemostatic alterations. An underestimated aspect of POCT is the importance of established quality management procedures. For this purpose control materials based on plasma or artificial fluids are being applied. In spite of often higher costs we appraise the use of POC analysis in many settings as justified because of the gain of time and the overall better process quality, i.e. targeted haemostasis therapy instead of consecutive application of different therapeutic options.

Gelatin solutions are often given in clinical practice once the maximal dose of a median-weight hydroxyethyl starch (HES) has been reached. Colloids are usually combined with lactated Ringer's solution (RL). Whether the combined administration of colloids and/or crystalloids affects blood coagulation is not known. We diluted blood by 20%, 40%, and 60% with RL, gelatin (Gelofusin), 6% HES 130/0.4 (Voluven), and 6% HES 200/0.5 (Iso-Hes), as well as with combinations of these solutions at a ratio of 1:1 (gelatin/RL, 6% HES 130/0.4:RL, 6% HES 200/0.5:RL, 6% HES 130/0.4:gelatin, 6% HES 200/0.5:gelatin). Thereafter, blood was analyzed by using modified thrombelastograph coagulation analysis (ROTEG) and clotting time, clot formation time, and maximal clot firmness were determined. RL had the least effect on hemostasis. Gelatin administered alone impaired the coagulation system significantly less than each median-weight HES administered alone. We conclude that gelatin combined with 6% HES 200/0.5 or 6% HES 130/0.4 decreases hemostasis6% HES 200/0.5 or 6% HES 130/0.4 administered alone.The effect of the combined administration of different colloids and/or crystalloids on coagulation is not known. We show that hemostasis is less impaired using a combination of gelatin and median-weight starches than using median-weight starches alone. Furthermore, the combination of lactated Ringer's solution and gelatin decreases the coagulation system to the same extent as the combination of lactated Ringer's solution and 6% hydroxyethyl starch 130/0.4.