ROTEM and multiplate a suitable tool for POC?

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1 STATE OF THE ART 4D-S27-02 ISBT Science Series (2010) 5, ª 2010 The Authors. Journal compilation ª 2010 International Society of Blood Transfusion ROTEM and multiplate a suitable tool for POC? A. Pape, C. F. Weber, P. Stein, & K. Zacharowski Clinic of Anesthesiology, Intensive Care Medicine and Pain Management, J.W. Goethe University Hospital, Frankfurt Main, Germany Introduction The early recognition of perioperative coagulation disorders is essential to identify non-surgical reasons of bleeding, to initiate appropriate haemostatic treatment and finally to reduce perioperative blood loss. Conventional laboratory coagulation tests include prothrombin time (PT), activated partial thromboplastin time (aptt), fibrinogen levels and platelet counts. Although these parameters are frequently assessed, their value has been challenged at least in the setting of acute perioperative bleeding [1]. PT and aptt are performed in an artificial in vitro system, i.e. in plasma separated from whole blood, which is then warmed to 37 C and buffered to a ph of 7Æ4. These standardized conditions often do not reflect the patient s situation [2]. Plasmatic in vitro tests merely reflect the time elapsing until the activation of thrombin, but provide only marginal information about the functional state of the coagulation system (e.g. platelet function, interaction of plasmatic coagulation factors with platelets and red blood cells, clot firmness, fibrinolysis). Routine coagulation tests are mostly performed in the central laboratory, i.e. remote from the operating theatre. As a consequence, test results are often not available in time or with a considerable delay [3]. Current devices designed for point-of-care (POC) monitoring of coagulation have been designed for the assessment of coagulation directly at the bedside, so that results could be available earlier. Moreover, POC tests are usually performed in whole blood, thereby comprising interactions between cellular components (i.e. platelets and red blood cells) and plasmatic coagulation factors. Commonly used POC devices for assessment of plasmatic coagulation and platelet function are listed in Table 1. This review focuses on rotation thrombelastography (ROTEM Ò ) as a global coagulation test and multiple electrode aggregometry (MEA, Multiplate Ò ) for assessment of platelet function. Correspondence: Dr. med. Andreas Pape, Clinic of Anesthesiology, Intensive Care Medicine and Pain Management, J.W. Goethe University Hospital, Theodor Stern Kai 7, Frankfurt Main, Germany a.pape@em.uni-frankfurt.de Rotation thrombelastometry (ROTEM Ò ) The dynamic assessment of the blood clot s viscoelastic properties by means of thrombelastography was established by Hartert in This method allows the evaluation of global haemostatic function by continuous analysis of clot elasticity and fibrin viscosity, quantitative recording of the retraction process and fibrinolysis. Critical points of the clotting process can be identified on the basis of the trace recorded during clot formation and subsequent lysis [4]. While the terms Thrombelastograph Ò and TEG Ò were registered by Hemoscope Inc (Niles, IL, USA) in 1996, rotation thrombelastometry represents a modification of TEG Ò and the term ROTEM Ò was registered by Pentapharm (Munich, Germany) in Method Blood sample processing ROTEM Ò is performed in citrated whole blood, which is incubated at 37 C in a heated cup. A mobile pin is suspended into the blood sample and oscillates through an angle of 4Æ75. Rotation movements are detected by an optical unit consisting of a mirror plate at the end of the pin, a diode as a light source and a CCD chip as light sensor. While the pin rotates around the vertical axis, coagulation is initiated on the surface of the pin resulting in platelet activation and the formation of fibrin strands. The outer surface of the pin is getting linked with the inner surface of the cup, and the resulting impedance of the rotation movement reflects the firmness of the developing clot. Changes of the rotational amplitude over time are translated into a typical trace displaying clot firmness versus time (Fig. 1). The calculation of clot firmness is based on the assumption that an unimpeded rotation reflects a clot firmness of 0 mm and a complete blockade of rotation would correspond to a theoretical clot firmness of 100 mm [5]. Parameters and tests performed with ROTEM Ò ROTEM Ò analysis displays the dynamic of clot formation and lysis as well as the mechanic stability of the clot during these processes. Parameters assessed by ROTEM Ò are as follows: the time elapsed until initial fibrin formation (clotting 161

2 162 A. Pape et al. Table 1 Overview of POC tests of global plasmatic coagulation and platelet function Device Viscoelastic test devices Thrombelastograph (TEG) Sonoclot Analyzer Rotation Thrombelastometry (ROTEM) Platelet function test devices Platelet Function Analyzer (PFA-100) Rapid Plate Function Assay (RPFA, Verify Now) Platelet Works Clot firmness (mm) Multiple Electrode Aggregometer (Multiplate) a-angle ( ) Manufacturer Clotting time (CT) Time (min) Clot formation time (CFT) Hemoscope Corp., Niles, IL, USA Sienco Inc., Arvada, CO, USA Pentapharm GmbH, Munich, Germany Dade-Behring, Marburg, Germany Accumetrics, San Diego, CA, USA Helena Laboratories, Beaumont, TX, USA Dynabyte, Munich, Germany Maximum lysis (ML) Maximum clot firmness (MCF) Fig. 1 Schematic of ROTEM Ò test parameters. Clotting time (CT): time elapsed until initial fibrin formation (clotting time, CT); Clot formation time (CFT): time elapsed from initiation of clotting until a clot firmness of 20 mm is achieved, velocity of clot formation is also reflected by the slope of the tangent (a-angle); Maximum clot firmness (MCF): mechanic stability of the clot reflected by maximal amplitude of the trace; Clot lysis index (CLI): reduction of clot firmness after MCF in relation to MCF, maximum lysis (ML) is reflected by the inflection point of the curve. time, CT), the kinetics of fibrin formation and development of the definitive clot (a-angle and clot formation time, CFT), the mechanic stability of the fibrin clot (maximum clot firmness, MCF; amplitude of the trace after 5, 10, 20, 30 or 25 min, A5, A10, A20 or A30) and finally clot lysis reflected by index (CLI) and maximum lysis (ML) (cf. Table 2, Fig. 1) [1]. The ROTEM Ò system is equipped with an electronic pipette and has four independent channels, enabling the simultaneous performance of up to four different tests. Commonly used tests are ExTEM Ò and InTEM Ò as extrinsic and intrinsic screening tests and HepTEM Ò, ApTEM Ò 60 Table 2 ROTEM Ò tests and the reference ranges of their results [13] CT CFT a-angle A10 A20 A30 MCF CLI 30 ML Maximum Lysis (% of MCF) Clot lysis index after 30 min (% of MCF) Maximum clot firmness (mm) Amplitude after 30 min (mm) Amplitude after 20 min (mm) Amplitude after 10 min (mm) Slope of tangent at 2 mm Clot formation time (s) Clotting time (s) Activator ExTEM Ò Tissue factor ApTEM Ò Tissue factor + Aprotinin Under physiological conditions values comparable with ExTEM Ò. A better clot formation in ApTEM Ò compared with ExTEM Ò is an early sign of hyperfibrinolysis. FibTEM Ò Tissue factor + Cytochalasin D ND ND 9 25 ND ExTEMÒ amplitude low and FibTEMÒ amplitude normal: Fibrinogen level OK, Platelet deficiency ExTEM amplitude low, FibTEM amplitude normal: Fibrinogen deficiency InTEM Ò Partial thromboplastin phospholipids HepTEM Ò Partial thromboplastin Under physiological conditions values comparable with InTEM Ò. A better clot quality in HepTEM Ò compared with InTEM Ò indicates a heparin effect. phospholipids + Heparinase

3 ROTEM and multiplate 163 and FibTEM Ò as tests for heparin activity, hyperfibrinolysis and fibrinogen levels (cf. Table 2). Each test is initiated by recalcification of the blood sample and by adding activators of the extrinsic (tissue factor obtained from rabbit brain) or the intrinsic (partial thromboplastin phospholipids plus ellagic acid) pathway of the coagulation cascade. Amplitude and CFT of the ExTEM Ò and InTEM Ò traces are influenced by factor activities (ExTEM Ò : factors VII, X, II, I; InTEM Ò : factors XII, XI, IX, VII, X, V, II, I) as well as by platelets and fibrinogen levels. Fibrinogen is essential for clot stability: as the substrate of the coagulation system, fibrinogen is converted to fibrin monomers by thrombin and finally stabilized to insoluble fibrin polymers by factor XIII. In the case of an acute massive blood loss, fibrinogen is, however, the first factor to be affected by dilution kinetics and the first factor achieving its critical level [6,7]. An accepted standard for determination of fibrinogen levels is the von Clauss method, which is performed in diluted plasma samples titrated with excess thrombin. Using a standardized calibration curve, fibrinogen levels are calculated from the time elapsed until the onset of clot formation. However, in the presence of colloids (e.g. hydroxyethyl starch), this method may yield falsely high levels of fibrinogen [8 11]. Contrasting this, the FibTEM Ò test is not affected by interference with plasma substitutes. Basically, FibTEM Ò is activated in the same manner as the ExTEM Ò test. Additionally, the FibTEM Ò reagent contains cytochalasin D for inhibition of platelet aggregation, so that fibrinogen and factor XIII are the remaining active coagulatory agents [12] so that maximum clot firmness (MCF) assessed in this modified assay represents the level of functional fibrinogen [13,14]. ROTEM Ò also provides the possibility to detect hyperfibrinolytic states using the APTEM Ò test [5,15]. Similar to ExTEM Ò, the ApTEM Ò assay activates the extrinsic pathway. For inhibition of an assumed hyperfibrinolysis, the ApTEM Ò reagent contains aprotinin, so that ApTEM Ò serves as a negative control to ExTEM Ò. In the case of hyperfibrinolysis, pathological findings in ExTEM Ò include decreased MCF and premature clot lysis, while these results are not displayed in the ApTEM Ò trace. In the same manner, ROTEM Ò can detect heparininduced coagulation disorders using the HepTEM Ò test. Contrasting FibTEM Ò and ApTEM Ò, the HepTEM Ò assay activates the intrinsic pathway of the coagulation system and contains heparinase for elimination of heparin effects. In the case of a heparin-induced coagulopathy, CT would be prolonged in InTEM Ò (this effect would not be seen in ExTEM Ò, as only InTEM Ò is sensitive to heparin). If CT is prolonged both in InTEM Ò and HepTEM Ò, a heparin-related effect can be excluded and a factor deficiency is likely [12]. Perioperative application As described earlier, ROTEM Ò enables the assessment of (i) hyperfibrinolysis, (ii) fibrinogen deficiency, (iii) disorders of fibrin polymerization, (iv) deficit of plasmatic coagulation factors and or platelets and (v) heparin effects. The heparin effects are relevant in situations requiring full heparinization (e.g. cardiothoracic surgery). For POC monitoring of anticoagulation during cardiopulmonary bypass (CBP), heparinase-based assays appear at least as suitable as kaolin-activated clotting time (ACT) [4]. However, during full heparinization, heparinase-based assays can detect coagulation disorders not related to heparin [12]. Although heparin effects are neutralized with protamine at the end of CPB, coagulation may still be severely impaired by platelet dysfunction and hyperfibrinolysis, both elicited by contact activation of platelets and coagulation factors on the extrinsic surface of CBP system. In this situation, it is crucial to restore haemostatic function and thereby to limit perioperative blood loss. Some non-randomized trials suggest that viscoelastic tests appear superior to conventional coagulation tests regarding the prediction of perioperative bleeding in patients undergoing cardiac surgery [16,17]. It is generally accepted that sufficient haemostasis therapy can be established on the basis of POC test results [18]. Indeed, the implementation of transfusion algorithms based on the results of POC tests has repeatedly been demonstrated to reduce the need for allogenic blood transfusions in adults and children undergoing cardiac surgery [19 22], suggesting that this procedure might be cost-effective [23,24]. ROTEM Ò tests are also increasingly performed in patients undergoing orthotopic liver transplant (OLT). In these patients, coagulation is deranged because of decreased factor synthesis, impaired platelet function and by insufficient clearance of activated factors resulting in mild to moderate consumptive coagulopathy with hyperfibrinolysis. During OLT, additional impairment of coagulation occurs during the anhepatic phase and after organ reperfusion. Indeed, several authors described the use of viscoelastic POC tests during OLT [25 27] and their value in the assessment of hypocoagulable states during surgery as well as in the management of hypercoagulability and thrombotic complications in the postoperative phase. ROTEM Ò -guided haemostatic management has also been reported in the fields of trauma, obstetrics or sepsis [28 32]. In summary, most of these reports tended to use plasma fractions rather than fresh-frozen plasma (FFP). As the efficacy [33,34] and risk benefit ratio [35 38] of FFP transfusions have repeatedly been challenged, the supplemental or alternative use of plasma fractions might be advantageous. However, to which extent the indication for their use might actually be guided by POC monitoring or by

4 164 A. Pape et al. clinical judgement remains to be demonstrated in adequately powered prospective randomized clinical trials (RCTs). Limitations The major advantage of ROTEM Ò analysis is the rapid availability of results. Indeed, first results are available within min after beginning the measurement, but maximum clot firmness is usually achieved after min and the detection of a late hyperfibrinolysis might require test duration over a period of up to 60 min [26]. Therefore, POC tests might not be suitable for the initial management of a massive haemorrhage (e.g ml blood loss within 20 min [39]). In this situation, however, RO- TEM Ò analysis might be used to retrospectively confirm the appropriateness of coagulation management. Appropriate use of laboratory equipment requires maintenance, supervision and quality control on a regular basis. However, POC tests are usually run by non-laboratory personnel with a variable degree of training and expertise. To overcome these problems and to reduce the workload of medical personnel (e.g. anaesthesiologists, intensive care personnel), some hospitals have moved POC devices to the central laboratory, thereby improving diagnostic quality, but foiling the originally provided time advantage [1]. ROTEM Ò analyses display coagulatory function more comprehensively than conventional tests such as PT or aptt (test are performed at the patient s actual ph, sample comprises plasmatic coagulation factors and cellular components). However, blood samples are still warmed to 37 C, which may not reflect the hypothermic milieu of a massively bleeding patient. Moreover, ROTEM Ò tests are performed under static conditions in a cuvette void of endothelial cells, which exert significant influence on coagulatory function. Therefore, the results of ROTEM Ò tests should per se be interpreted with reference to the clinical aspect (e.g. diffuse bleeding at the surgical site). ROTEM Ò analyses are not apt for the detection of disorders in primary haemostasis like von Willebrand s syndrome or the effect of antiplatelet drugs (acetylsalicylic acid, clopidogrel, glycoprotein IIb-IIIa inhibitors), as these effects are overwhelmed by thrombin generation within the test assays. Finally, there is only little correlation between PT INR (international normalised ratio) and CT in the ExTEM Ò test, so that ROTEM Ò is not an appropriate tool for monitoring the effects of oral anticoagulants (e.g. Cumarine). Assessment of platelet function: MEA (Multiplate Ò ) MEA (Multiplate Ò, Dynabite, Munich, Germany) was introduced in 2005 and represents a novel device for POC assessment of platelet function. Multiplate Ò operates on the basis of whole blood impedance aggregometry [40], which was established by Cardinal and Flower in 1979 [41]. Similar to ROTEM Ò, the complexity of the Multiplate Ò device is moderate, so that MEA appears attractive for POC use [42]. Method Sample processing MEA is performed in whole blood (temperature 37 C) anticoagulated with heparin or hirudin. The use of hirudin enables optimal reproducibility of test results. Analysis should be performed within min after sample drawing [43]. Impedance aggregometry is based on the detection of changes in electrical impedance caused by accretion of platelets on the surface of electrodes. The latter process requires activation of platelets, which is realized with several specific test solutions. The test cell of the Multiplate Ò device consists of two pairs of silver-coated conductive copper wires suspended into the blood sample reagent mixture, which is automatically stirred at 800 U min with a disposable magnetic PTFE-coated stir bar. The continuously recorded impedance between the two wires is proportional to the number of platelets adhering to the electrodes. Dynamic changes in impedance are translated into arbitrary aggregation units (AU) and are typically plotted versus time (Fig. 2). Sufficient testing quality is assumed, when the difference between the curve is <20%. Parameters and tests Aside from AU, quantitative analysis yields velocity of aggregation (slope of trace, AU min) and the area under Fig. 2 Typical plot of arbitrary aggregation units (AU) versus time in two Multiplate Ò tests. The area under the curve (AUC = AU*min) has the highest diagnostic power and is affected by the total height of the aggregation curve as well as by its slope. The latter reflects the velocity of the aggregation process. The results of the tests are mean values of the parameters determined with each curve.

5 ROTEM and multiplate 165 Table 3 Multiplate Ò tests including their activators, investigated effects and sensitivity Test Activator Effect Sensitive for Not sensitive for COL-test Ò Collagen Release of arachidonic acid Acetylsalicylic acid GP IIb IIIa inhibitors Clopidogrel von Willebrand s syndrome ASPI-test Ò Arachidonic acid Conversion to TXA2 by COX Acetylsalicylic acid GP IIb IIIa inhibitors Clopidogrel von Willebrand s syndrome ADP-test Ò Adenosine diphosphate Stimulation of ADP receptors Clopidogrel GP IIb IIIa inhibitors Acetylsalicylic acid von Willebrand s syndrome TRAP-test Ò TRAP-6 Stimulation of thrombin receptors GP IIb IIIa inhibitors Clopidogrel von Willebrand s syndrome RISTO-test Ò Ristocetin vwf dependent platelet activation (via GP Ib receptor) Severe von Willebrand s syndrome Bernard Soulier s syndrome Acetylsalicylic acid Mild von Willebrand s syndrome curve (AUC, AU*min). Among these parameters, AUC has the strongest diagnostic power [40]. The Multiplate Ò device has five channels, allowing the simultaneous performance of five tests (Table 3): COL-test Ò : Assessment of collagen-induced activation. The GP Ia IIa receptor is activated by addition of collagen, resulting in liberation of arachidonic acid, release of thromboxan A2 (TXA2) and platelet activation. Prerequisite is a sufficient activity of cyclooxygenase (COX). ASPI-test Ò : Evaluation of the effects of acetylsalicylic acid and NSAIDs on COX-dependent aggregation. The ASPI-test Ò is initiated by adding arachidonic acid, the substrate of COX. In the case of acetylsalicylic acidinduced COX inhibition, arachidonic acid is not metabolized to TXA2, and platelets cannot be activated by this test. ADP-test Ò : Sensitive to the effects of ADP antagonists (i.e. clopidogrel). In the presence of these drugs, platelet activation via ADP receptors is impossible. TRAP-test Ò : Platelets are activated by adding thrombin receptor activating peptide 6 (TRAP-6) via thrombin receptors PAR1 and PAR4. This test is sensitive to the effects of GP IIb IIIa antagonists. RISTO-test Ò : By addition of ristocetin, this test assays aggregation processes depending on von Willebrand factor (vwf) and or GP Ib. Perioperative application Only very few publications reporting the perioperative use of Multiplate Ò are available up to date. However, MEA has already been used for monitoring the effects of antiplatelet drugs (acetylsalicylic acid, clopidogrel, GP IIb IIIa anatagonists, ADP receptor blockers) in the field of cardiology [44 47] or neurology [48]. Moreover, the effects of non-opioid analgesics [49], body core temperature [50,51], cardiopulmonary bypass [52] or the infusion of colloidal solutions [53,54] on platelet aggregation were studied by use of MEA. Bergman et al. reported the use of MEA for guiding the removal of an epidural catheter in a patient requiring dual platelet inhibition (acetylsalicylic acid plus clopidogrel) [55]. Weber et al. used MEA for monitoring the effects of 1-desamino-8-D-arginine vasopressin (DDAVP) for the treatment of COX inhibitor related platelet inhibition [56] and of CPB-related impairment of platelet aggregation in 58 patients [57]. Rahe-Meyer et al. demonstrated that MEA might have a predictive value regarding platelet transfusion requirements during and after cardiac surgery (n = 60 patients) [58], while Poston et al. found that MEA combined with thrombelastography could be predictive regarding blood loss as well as thrombotic complications after off-pump coronary arterial bypass surgery (n = 76 patients) [59]. Limitations Because of the novelty of the device, only very few data about the perioperative use of Multiplate Ò are presently available. While impaired platelet function as detected by MEA was associated with increased transfusion rates in smaller non-randomized studies, the predictive value of MEA regarding perioperative blood loss and transfusion requirements still remains to be validated in RCTs. Inasmuch, the potential relevance of MEA in POC-guided transfusion algorithms cannot finally be estimated at the present time point. Like ROTEM Ò, the performance of Multiplate Ò tests requires 20 min. Moreover, a resting time of 30 min after blood sampling is recommended before testing [45], which may impede the immediate detection of platelet dysfunction in the perioperative setting. Limitations related to personnel requirements are identical with ROTEM Ò. While the RISTO-test Ò may be sensitive for severe forms of von Willebrand s syndrome or Bernard Soulier syndrome [60], only very few data exist about the sensitivity of Multiplate towards disorders of primary haemostasis. Finally, the results of Multiplate analysis correlate significantly with platelet number and haematocrit [61,62].

6 166 A. Pape et al. Summary and conclusion POC monitoring of coagulation can be helpful to early recognize non-surgical reasons of bleeding and to initiate a targeted treatment of several coagulation disorders. In contrast to conventional coagulation tests (PT, aptt, platelet counts), POC tests such as ROTEM Ò or Multiplate Ò are performed in whole blood, which comprises interactions between cellular components (platelets, red blood cells) and plasmatic coagulation factors. ROTEM Ò provides screening tests for the intrinsic and extrinsic pathway of the coagulation cascade as well as specific tests for heparin effects, fibrin polymerization and hyperfibrinolysis and is therefore increasingly used in clinical situations with inherent risk of coagulation disorders: i) dilutional coagulopathy in massive haemorrhage, ii) assessment of hypo- or hypercoagulable states and iii) hyperfibrinolysis in cardiac surgery, orthotopic liver transplant, sepsis, trauma, urology and gynaecology obstetrics. Multiplate Ò tests enable differentiated analysis of platelet activation and provide information about the global state of platelet function as well as about the extent of a possible platelet inhibition (acetylsalicylic acid, clopidogrel, GP IIb IIIa inhibitors). Multiplate Ò can detect severe forms of von Willebrand s syndrome and platelet disorders. The validity of Multiplate Ò tests depends, however, on platelet count and haematocrit. ROTEM Ò alone or in combination with Multiplate Ò appears to be predictive of blood loss as well as thrombotic complications, which particularly appears attractive in cardiovascular surgery. Indeed, retrospective studies indicate that the institution of POC-based transfusion algorithms might reduce perioperative blood transfusion rate and hence transfusion associated costs. However, the superiority of POC guided to conventional coagulation management still needs to be demonstrated in prospective RCTs. POC systems have been designed for coagulation analysis directly at the bedside, which may help to gain time in for therapeutic decisions. However, also POC tests require time, so that their value may be limited in situations of profuse bleeding. Moreover, POC tests require personnel resources for test performance as well as for maintenance, supervision and quality assurance. In summary, ROTEM Ò and Multiplate Ò are suitable tools for POC inasmuch as they identify concise therapeutic targets and may thereby enable an effective coagulation management. Their value needs, however, to be corroborated with evidence from adequate prospective RCTs. Generally, the appropriate use of POC monitoring in the clinical routine requires strict quality controls and trained personnel to ensure optimal accuracy and performance. Disclosures None. References 1 Ganter MT, Hofer CK: Coagulation monitoring: current techniques and clinical use of viscoelastic point-of-care coagulation devices. Anesth Analg 2008; 106: Tieu BH, Holcomb JB, Schreiber MA: Coagulopathy: its pathophysiology and treatment in the injured patient. World J Surg 2007; 31: Kozek-Langenecker S: Management of massive operative blood loss. 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