Veterinary Clinical Pathology ISSN 0275-6382

REVIEW

Viscoelastic coagulation testing: technology, applications, and limitations

Maureen A. McMichael1, Stephanie A. Smith2
1

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, and 2Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA

Key Words Hypercoagulability, ROTEM, Sonoclot, thrombelastography, thromboelastometry Correspondence Maureen A. McMichael, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 1008 West Hazelwood Dr., Urbana, IL 61802, USA E-mail: mmcm@illinois.edu DOI:10.1111/j.1939-165X.2011.00302.x

Abstract: Use of viscoelastic point-of-care (POC) coagulation instrumentation is relatively new to veterinary medicine. In human medicine, this technology has recently undergone resurgence owing to its capacity to detect hypercoagulability. The lack of sensitive tests for detecting hypercoagulable states, along with our current understanding of in vivo coagulation, highlights the deciencies of standard coagulation tests, such as prothrombin and partial thromboplastin times, which are performed on platelet-poor plasma. Viscoelastic coagulation analyzers can provide an assessment of global coagulation, from the beginning of clot formation to brinolysis, utilizing whole blood. In people, use of this technology has been reported to improve management of hemostasis during surgery and decrease usage of blood products and is being used as a rapid screen for hypercoagulability. In veterinary medicine, clinical use of viscoelastic technology has been reported in dogs, cats, foals, and adult horses. This article will provide an overview of the technology, reagents and assays, applications in human and veterinary medicine, and limitations of the 3 viscoelastic POC analyzers in clinical use.
VI. VII. Conclusions References

I. II.

III.

IV.

V.

Introduction Sonoclot A. Technology B. Variables C. Reagents and assays D. Application: human medicine E. Application: veterinary medicine F. Advantages and limitations Thrombelastography A. Technology B. Variables C. Reagents and assays D. Application: human medicine E. Application: veterinary medicine F. Advantages and limitations Thromboelastometry A. Technology B. Variables C. Reagents and assays D. Application: human medicine E. Application: veterinary medicine F. Advantages and limitations Viscoelastic testing A. Advantages B. Limitations C. Comparison with standard plasma-based coagulation testing D. Methods

Introduction
Hemostasis is a complex series of physiologic events culminating in the formation of a brin clot through the proteolytic action of thrombin on brinogen. Recent advancements in the study of coagulation have elucidated the important contribution of cells to the hemostatic process. The cell-based model places emphasis on platelets and tissue factor-bearing cells while also taking into account the contribution of membrane surfaces, microparticles, enzyme systems, and endothelial cells. An in-depth review of the cell-based model of coagulation has been published previously.1 Our current understanding of in vivo coagulation highlights the limitations of standard coagulation tests, such as prothrombin time (PT) and activated partial thromboplastin time (aPTT), which do not incorporate cellular elements or only provide data on isolated components of the coagulation cascade. Although valuable

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for examining specic components of enzymatic cascades, these tests overlook such factors as rate of clot formation, overall clot strength, and rate and degree of dissolution, factors that represent signicant interactions essential to evaluation of the hemostatic system in clinical patients. In 1889 Hayem2 suggested that quantication of the changes that occur in blood viscosity during clotting could be utilized as the basis for a test that monitors coagulation function. Blood clots have both elastic and viscous properties and technological methods have been developed to assess these properties. Elasticity, or rmness, refers to the reversible deformation of a material under stress; the modulus of elasticity is a measure of stiffness. Viscous properties, referring to stickiness or thickness, cause blood to have high resistance to ow. Elasticity of a blood clot is affected primarily by brin and platelets in the sample.35 The rst coaguloviscometer, a machine designed to detect viscous changes in blood during clotting, was introduced in 1910 by Koffman.6 Signicant improvements have occurred in the evolution of the technology since that time. Viscoelastic point-of-care (POC) devices provide in vitro assessment of global coagulation, from beginning of clot formation to brinolysis. Most conventional coagulation tests end when the rst brin strands are developing, whereas viscoelastic coagulation tests begin at this point and continue through clot development, retraction, and lysis. An analogy, suggested by Hartert,7 compares coagulation testing to the building of a house; conventional coagulation tests end with the laying of the foundation, whereas viscoelastic testing provides information about the entire house, including the speed of the building process and the nal strength of the completed house. This technology measures the kinetics of clot formation (the time needed for the clot to form), the mechanical properties of the clot (tensile strength), and the time to dissolution of the clot (brinolysis). The tensile strength of the clot provides information about the capacity of the clot to achieve hemostasis, and the kinetics determine the adequacy of the quantitative factors available for the clot to form. The capability of utilizing whole blood in viscoelastic kinetic testing is an additional advantage of this approach for evaluating the coagulation system. Whole blood contains cells that provide the charged phospholipid cell surfaces necessary for enzymatic reactions. It also provides the platelets that further participate in coagulation by releasing granule contents and providing a surface for amplication and propagation of the clot.1 Clinically relevant benets of viscoelastic testing of clot dynamics are found in both human and veterinary

medicine. The use of viscoelastic coagulation analyzers has resulted in improved management of hemostasis during surgery, decreased usage of blood products, rapid identication of mechanical (vessel) bleeding postoperatively, more accurate anticoagulation management, and rapid screening for hypercoagulability.810 This article will review the technology, reagents, applications, and limitations of the viscoelastic POC analyzers available for clinical use. There are 3 instruments currently used in veterinary and human medicine; the Sonoclot coagulation and platelet function analyzer or Sonoclot (Sienco Inc., Arvada, CO, USA), the TEG thrombelastograph hemostasis analyzer or TEG (Haemonetics Corporation, Braintree, MA, USA), and the ROTEM (Pentapharm GmbH, Munich, Germany). The Sonoclot and ROTEM measure changes in impedance to movement of a vibrating probe immersed in a blood sample, whereas TEG utilizes an oscillating cup with a xed probe or piston. The probe is a torsion wire in TEG technology, whereas an optical detector is used by the ROTEM. All 3 instruments measure the rate of brin formation, clot strength, and clot lysis. The TEG and ROTEM have become increasingly used in POC management of trauma and perioperative bleeding during cardiac and liver transplantation in people.8,11,12 In 1966 the Haemoscope Corporation (now part of Haemonectics Corporation) registered trademarks for the terms thrombelastography and TEG; thus, these terms are limited to evaluations done with the Haemoscope analyzers. Pentapharm GmbH has also registered trademarks for the terms ROTEM and thromboelastometry. The technology and data obtained are similar; for the purposes of this review TE will refer to analysis done with either the ROTEM or the TEG. The variables recorded by the 3 instruments and the associated terminology are summarized (Table 1).

Sonoclot
The Sonoclot analyzer, introduced by von Kaulla in 1975,13 uses whole blood or plasma. The instrument detects viscoelastic changes that occur during clotting (http://www.sienco.com/sonooverview.html). Technology The procedure begins with a hollow, disposable plastic probe placed onto the transducer head before adding the test sample, either whole blood or plasma, to a cuvette. The sample is mixed automatically and then the probe is immersed into the sample and begins to oscillate. When the clot begins to form it impedes the movement of the probe by creating a viscous

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Table 1. Comparison of the coagulation variables recorded by the Sonoclot, TEG, and ROTEM. Development of Clot Initial brin formation Development of clot or rapidity of clot formation Maximal clot strength Fibrinolysis Factors Affecting Clot Factor XII and XI activity; reective of intrinsic pathway if activators not used Factor II and VIII activity, platelet count and function, thrombin, brinogen, HCT Fibrinogen, platelet count and function, thrombin, factor XIII activity, HCT Hyperbrinolysis Sonoclot SonACT Clot rate (CR) TEG Reaction time (R) Kinetics (K) and a angle (a) ROTEM Clot time (CT)

Clot formation time (CFT) and a angle (a) Peak amplitude Maximum amplitude Maximum clot and time to peak (MA) rmness (MCF) R3 Clot lysis (CL30, Lysis (LY30, LY60) CL60)

drag, and this impedance is captured electronically as resistance to motion that the probe encounters in the blood sample.14 Variables The Sonoclot analyzer provides information both quantitatively and qualitatively as Sonoclot Signature (SS; Figure 1). Coagulation reactions develop from the beginning of the SS and continue throughout the liquid phase. The end of the liquid phase is reported as Onset in the SS (also called the SonACT) and is dened as an upward deection of 1.0 mm calculated by the instrument.14 The gradient of the rst slope (R1) indicates the initial rate of brin formation. This is the point where identication of hypercoagulability is made and is the reference point for anticoagulant management. There is a variable shoulder between R1 and R2 that represents the lag time before the start of contraction of the brin strands by the action of platelets. The second

Figure 1. Sonoclot Signature. Shown are activated clotting time (SonACT), clot rate, time to peak, clot retraction, and the 3 slopes, R1, R2, and R3. ACT, activated clot time.

slope (R2) indicates platelet action resulting in contraction of the clot, further brin formation, and brin polymerization. The third slope (R3) is a downward slope indicating platelet retraction and the clot pulling away from the cuvette walls. R3 is an indicator of platelet number and function. As a normal clot retracts it tightens and causes the SS to rise owing to increased impedance of the probe to movement. Then at some later time, the SS falls when the clot pulls away from the inner surface of the probe or cuvette owing to loss of impedance, allowing free movement of the probe. Measurements of the time it takes for retraction and the degree of retraction permit analysis of platelet function. Coagulation and brin gel formation are not affected by hyperbrinolysis or early clot breakdown, but platelet function can be signicantly reduced in these circumstances owing to inhibition of platelet function by plasmin. If poor clot retraction is the result of plasmin inhibition of platelets, treatment of hyperbrinolysis with plasmin inhibitor will improve the abnormal clot retraction as observed on the SS. In this case, the SS will lack the characteristic rise (R2) and fall (R3) associated with normal clot retraction owing to plasmin inhibition of platelet function. Quantitative results are reported as activated clot time (ACT) in seconds (s), clot rate (CR; D signal/s), platelet function (PF; no units), time to peak (TP; s), and peak clot strength (PCS; clot signal). ACT is comparable to conventional plasma ACT.14 CR represents the rate of clot formation and is the maximum slope of the SS. PF is derived from the timing and quality of clot retraction, is calculated using an algorithm performed by the accompanying software program, and represents platelet function.15 TP is used to characterize clot retraction, and faster TP times are correlated with greater platelet function. PCS is the point in the SS with the largest signal amplitude and represents maximal clot stiffness. In people PCS is inuenced by brinogen concentration.

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Reagents and assays Cuvettes contain different coagulation activators or inhibitors depending on the type of analysis desired. A cuvette without activator is frequently used to create the initial SS (Figure 1). Celite, a contact activator, is available in high concentration to measure ACT and low concentration to accelerate analysis and creation of the SS. Glass beads heparinase are also available to measure coagulation in the absence and presence of heparin. Application: human medicine One study comparing the Sonoclot with conventional ACT instruments (eg, Hemochron; ITC, Edison, NJ, USA) found the 2 instruments to be comparable.16 Use of the Sonoclot analyzer has been reported during cardiopulmonary bypass and liver transplant surgery in people as a rapid method to monitor anticoagulant therapy (Figure 2).10,17 It has also been used to identify hypercoagulability and to evaluate platelet function in high-risk human populations.18,19 The effects of specic anticoagulants, such as heparin, can be monitored using the Sonoclot,20 and it is also being used to evaluate newer anticoagulants in human medicine.20 Application: veterinary medicine In veterinary medicine, reports describing use of the Sonoclot are limited. Normal values have been reported in horses and foals, with foals demonstrating increased PF compared with adult horses.21 Sonoclot values measured in critically ill foals were evaluated for prognostic capabilities. Foals with decreased CR or slow clot formation on admission were more likely to be euthanized or die.15 Advantages and limitations The Sonoclot is reported to have better interoperator reliability compared with the TEG.14 Sonoclot results

have been shown to be inuenced by several variables, including age, sex, and platelet count.22 Additional studies showed poor reproducibility of some of the measured variables, especially CR and PF.2327 Other studies, however, have reported good reliability in patients undergoing cardiac surgery.25,26 In one study, precision for the Sonoclot was similar to that for TE.27 The Sonoclot has only 1 cuvette and does not permit duplicate samples to be run simultaneously. In addition, there does not appear to be a standardized protocol for new operators to adopt. Reports on the use of the Sonoclot use different activators, variable periods of warming, and variable holding times, and some use fresh whole blood whereas others used whole blood.15,16,20,28 The Sonoclot is the least expensive of the 3 POC coagulation instruments.

Thrombelastography
Thrombelastography was rst described by Hartert in 1948.29 Similar to the Sonoclot, TE assesses the viscoelastic properties of whole blood under low shear conditions and provides information about global hemostatic function from the beginning of clot formation through clot retraction and brinolysis (http:// www.haemoscope.com/technology/index.html). Technology The TEG measures the physical properties of the clot via a cylindrical cup heated to 371C that oscillates in 10-second cycles. A pin held by a torsion wire is suspended in the cup and is monitored for motion. As the clot starts to form, brin strands develop between the pin and the inner wall of the cup. This results in torque on the immersed pin that is transmitted to the torsion wire and converted to an electrical signal. As clot lysis occurs the bonds between the pin and clot are broken, resulting in decreased movement of the pin. A mechanical electrical transducer converts the rotation movement of the pin to an electrical signal that is

Figure 2. Abnormal Sonoclot Signature. (A) Part of a tracing before heparin administration. (B) Tracing after administration of heparin, demonstrating later onset, lower clot rate, and lack of clot retraction.

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Figure 3. Schematic of thromboelastometry (ROTEM; top) and thrombelastography (TEG, bottom). ROTEM gure shows clot time (CT), clot formation time (CFT), a angle (a), and maximum clot rmness (MCF). TEG gure shows reaction time (R), clot formation (K), a angle (a), and maximum amplitude (MA).

displayed as a TEG tracing (Figure 3). There are 2 slots for cups that can be run simultaneously for duplicate sampling or with different reagents. Variables A graphical tracing of viscoelastic changes displays initial brin formation (reaction time, R), kinetics of brin formation and development of the clot (K and a angle), and maximal strength of the brin clot (maximum amplitude [MA]) (Figure 3). Two additional measurements representing brinolysis or clot lysis at 30 and 60 minutes (CL30, CL60) indicate clot stability and are frequently absent from published tracings owing to the time required for normal brinolysis to occur. Reaction time can be expressed in 2 equivalent ways as distance in mm or time in minutes. The chart speed of the TEG is 2 mm/min; thus, time in minutes is equal to the distance in mm divided by 2. In earlier reports of studies using noncitrated blood R was dened as the time from initiation of the test to the point where the curve is 1 mm wide.30 Later, investigators suggested using the point where the curve is 2 mm wide for citrated blood, and this is the most commonly cited measurement for R today.3134 The time at which R is measured is the point at which standard plasmabased clotting assays would end. Prolonged R is seen with deciencies in coagulation factors. K and a angle (a) measure the rapidity of clot development from the beginning of the visible phase of coagulation to a dened level of clot strength. K is the time from initiation of clotting (2 mm) until an amplitude of 20 mm is reached and is measured in seconds. This aspect of the tracing is thought to be affected by activities of factors (F)II and VIII, platelet count and function, thrombin and brinogen concen-

trations, and HCT35,36 (http://www.haemoscope.com/ technology/index.html). MA represents the maximal strength of the brin clot and is recorded as the maximal width of the divergence of the lines on the tracing. It indicates global clot strength and is affected by brinogen, platelet count and function, thrombin, FXIII, and HCT.35,36 It is measured as the difference in mm above baseline on the tracing. Schematics frequently depicts the MA with a double-headed arrow that goes both above and below the baseline; although not technically correct, this is most likely an attempt to help the reader visualize how MA reects overall clot strength. Clot lysis, represented by CL30 and CL60, indicates % lysis that has occurred at 30 and 60 minutes, respectively, after MA has been reached and indicates clot stability. High CL percentages correspond with rapid brinolysis or platelet contraction. Other analyses that have been reported utilizing the TEG include TEG-index, coagulation index (CI), G (global clot strength), and total thrombin generation (TTG). TEG-index was developed to compare celiteactivated whole blood with nonactivated (native) whole blood from the same patient using the TEG.37 Celite activation of blood from normal patients had a much faster onset of coagulation, faster clot rate, and greater clot strength compared with native blood. The theory was that inherent hypercoagulability in native blood would nullify the differences between native and celite-induced TE, reecting in vivo hypercoagulability in abnormal individuals. Statistically signicant differences between the 2 types of tracings were used to develop an equation based on discriminant analysis between people with normal hemostasis and cancer patients.37 A higher TEG-index representing hypercoagulability was found in 98.9% of cancer patients in the study.37,38 CI is a computer-calculated linear combination of R, K, MA, and a angle and is thought to provide an index of coagulation in one simple number, rather than having to interpret all TEG variables.39 Adding all the TE values into a single number, while simplifying interpretation, signicantly diminishes the capabilities of this technology to discriminate hemostatic components, such as platelet function, factor levels, and brinolysis. Representing shear elastic modulus, G has also been reported.40,41 This value is reported to represent global coagulation in a single output number derived from the following formula: G = 5000 MA/(100 MA). Note that G is dependent only on MA; thus, like MA it is a function of brinogen concentration, platelet count and function, thrombin concentration, FXIII activity, and HCT. G increases exponentially compared

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with MA, which permits more sensitive resolution at high amplitudes. TTG is calculated by measuring the area under the thrombus velocity curve (dynes/cm2) and has been shown to correlate with thrombinantithrombin complex analysis, which is a marker of the amount of thrombin generated.42 PlateletMapping is a trademarked TEG application that measures % inhibition of platelet function compared with maximal uninhibited platelet function. It compares a tracing obtained when brinogen is cleaved and cross-linked by reptilase and FXIIIa and both thrombin and platelets are inhibited (representing brin formation without any contribution from platelets) with that obtained after addition of platelet agonists (when only thrombin is inhibited). The data are adjusted based on a tracing obtained from whole blood without any inhibitors (representing platelet and brin function). The difference is a specic representation of platelet function.43 Reagents and assays Viscoelastic technology was originally designed as a POC monitoring method using native whole blood run within minutes of collection. In the laboratory setting, this approach is not practical; thus, citrated samples are frequently used. A comparison study showed that TEG measurements are not directly comparable between native and citrated samples.44 Most tests are run either using fresh whole blood or citrated whole blood; platelet-poor plasma may also be used. When citrated samples are used, delay in testing results in generation of FXIIa, which is not calcium-dependent and, therefore, not inhibited by citrate. As a consequence, recalcication of a citrated sample results in thrombin generation primarily as a function of FXIIa generation during storage before recalcication rather than as a function of the constituents present at the time of sample collection.45 Activators of coagulation available for use with the TEG include kaolin, platelet mapping system reagents, and a new RapidTEG reagent that contains tissue factor and kaolin. Activation of intrinsic or extrinsic pathways can be achieved with kaolin or tissue factor, respectively. Cups that contain heparinase are available from the manufacturer to permit identication of nonheparin-dependent coagulation abnormalities in the presence of heparin. Application: human medicine The use of TEG analysis in a variety of clinical settings has been reported in both human and veterinary medicine (Figure 4). In human medicine TE has appli-

Figure 4. Abnormal TEG tracings. The center tracing is from a normal individual. Innermost tracing indicates a hypocoagulable state: R and K are prolonged, and a and maximum amplitude (MA) are decreased. Outermost tracing indicates a hypercoagulable state: R and K are shortened, and a and MA are increased. Refer to Figure 3 for explanations of tracings and abbreviations.

cability in managing trauma and surgical patients, monitoring heparin therapy, and thrombophilia. TEG has been used to assess the coagulation status and to predict early transfusion requirements of trauma patients.46 Tissue factor-activated TEG, marketed as RapidTEG, is being used in emergency rooms as an early diagnostic tool in trauma patients with suspected coagulopathies.47 Recently, TEG was reported to be more sensitive than plasma-based tests (eg, PT and aPTT) in detecting hypercoagulable states in trauma patients.48 TEG has also been repeatedly applied to human surgical populations. In one study of patients who had undergone cardiopulmonary bypass surgery, postoperative bleeding was more accurately predicted by TEG (87%) than conventional coagulation tests (51%) or ACT (30%).49 TEG has been used to guide FVIIa therapy in surgical patients,50 to monitor hemostasis during liver transplantation51 and cardiac surgery, and to limit unnecessary use of blood components.52 TEG has been used to predict the need for antibrinolytic therapy in patients receiving liver transplant.53 The MA recorded by TEG has been shown to be predictive of postoperative thrombotic complications in surgical patients.54 Heparinase has been reported for use with TEG permitting identication of abnormal coagulation in heparinized patients.55 In one study TEG was able to detect clot dissolution before changes were seen in plasma brinogen concentrations.56 Application: veterinary medicine In animals normal TEG values have been reported for healthy dogs57 and, more specically, for normal Greyhounds,40 horses,58 and cats.59 Canine reference intervals for kaolin-activated TEG have been reported.60 TEG results have also been described for dogs with hemostatic disorders,41 disseminated intravascular

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coagulation,33 parvoviral enteritis,61 neoplasia,62 and immune-mediated hemolytic anemia (IMHA)63 and for dogs admitted to an intensive care unit (ICU).64 In the parvovirus and DIC studies, TEG identied hypercoagulability in the patient populations. In a study of 27 dogs admitted to an ICU compared with 31 normal dogs, TEG results were abnormal in 52% of the ill dogs.64 Dogs in hypercoagulable states had signicant increases in D-dimer and brinogen concentrations, and there were signicant correlations between MA and brinogen concentration and between R and PT in the dogs studied.64 In a population of retired racing Greyhounds, all values except R and CL60 were signicantly different compared with those in nonGreyhound dogs.40 TEG results from Greyhounds indicated hypocoagulable states, which may provide a rationale for the increased tendency of this breed to bleed postoperatively. Conventional coagulation tests (PT, aPTT, ACT, and D-dimer) and TEG performed on multiple samples from the same group of dogs once weekly over 5 consecutive weeks were compared; a high degree of variability in the standard coagulation tests compared with TEG measurements was found.65 The capacity of tissue factor-activated TEG to identify hemostatic alterations in canine whole blood with varying dosages of low-molecular-weight heparin (LMWH) has been studied and found to be a promising new method for clinical evaluation of LMWHdosing in dogs.66 The effect of LMWH on hemostasis has also been reported in cats using TEG.67 Using TEG hypercoagulability was identied in healthy Beagle dogs after administration of prednisone, and PlateletMapping has been used to detect platelet inhibition by clopidogrel.68,69 Advantages and limitations In people a hypercoagulable state has been dened as the presence of at least 2 of the following: shortened R, increased a, or increased MA.46 As denitions have not been established yet for animals, comparisons among published reports are challenging. Studies have used different samples, including noncitrated whole blood and citrated and recalcied blood, various intrinsic and extrinsic activators, and different times from collection to performing TEG anlaysis. Owing to the lack of specic method details in published reports, critical evaluation of results obtained with this technology is difcult. The TEG system is less costly to purchase than the ROTEM, but provides only 2 channels. Use of this equipment for human patients has been approved by the US Food and Drug Administration (FDA).

Thromboelastometry
Rotational thrombelastography, termed thromboelastometry, also assesses the viscoelastic properties of whole blood under low shear conditions. The ROTEM is a modication of classic thrombelastography. Like the TEG, the ROTEM provides information about global hemostatic function from the beginning of clot formation through clot retraction and brinolysis (http://www.rotem.de/site/index). Technology The technical aspects of the ROTEM are slightly different from those of the TEG. There are 4 cylindrical cups and an optical detector system that detects the signal of a pin suspended in the blood sample cup. The cup is stationary and the pin oscillates. As brin forms between the cup and the pin, the impedance of the rotation of the pin is detected. As the blood clots the extent of the pins oscillation is reduced; this is measured by the angle of deection of a beam of light directed at the pin/wire transduction system. An ROTEM graphical tracing is recorded (Figure 3). The 4 channels can be used simultaneously allowing multiple specimens to be sampled. In addition, the ROTEM is equipped with an electronic pipette that permits consistency of dispensing the sample. Variables Graphical displays of viscoelastic changes indicate initial brin formation (clotting time, CT), the kinetics of brin formation and development of the clot (clot formation time [CFT]; a angle), the maximum strength of the brin clot (maximum clot rmness [MCF]), and brinolysis at 30 and 60 minutes (clot lysis, LY30, LY60). CT describes the time to initial brin formation and is an indicator of plasma coagulation factor activity. CFT corresponds to initial activation of platelets and brinogen; a corresponds to the slope of the tangent and describes the kinetics of clot formation. MCF is the maximal amplitude in mm above baseline reached during the test; MCF corresponds to the maximal clot strength and depends on both platelet and brinogen activation and the function of FXIII. Other values that can be calculated using ROTEM measurements have been reported and include maximum clot elasticity (MCE), global clot strength (G), and a version of TTG focusing on derivative curves. MCE is a calculated value derived from the MCF: MCE = (100 MCF)/(100 MCF). G is calculated using an equation identical to the one reported with the TEG, with MCF replacing MA: G = (5000 MCF)/

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(100 MCF). An alternative method of data analysis, somewhat comparable to calculation of TTG, utilizes software developed to analyze the rst derivative measurements of whole blood clot formation.70 The software produces a velocity prole consisting of maximum velocity (MaxVel) and time to MaxVel. The area under the velocity curve is also reported and is thought to be a measure of G.70 Reagents and assays Proprietary reagents available for use with the ROTEM include INTEM (strong intrinsic pathway activation), HEPTEM (strong intrinsic activation of coagulation in the presence of heparinase to neutralize effects of heparin in the sample), EXTEM (strong extrinsic pathway activation), FIBTEM (strong extrinsic activation of coagulation in the presence of cytochalasin D, a platelet inhibitor), APTEM (strong extrinsic activation of coagulation in the presence of aprotinin, a brinolysis inhibitor), and ECATEM (direct activation of coagulation with a snake-derived prothrombin activator). In order to attribute an abnormal result to platelet or brinogen abnormalities, a combination of EXTEM and FIBTEM may be used. These reagents permit specic evaluation of various components of the coagulation system. Application: human medicine The ROTEM has been applied to a variety of clinical conditions in human patients and is FDA-approved. Reference intervals have been established for all values, and the ROTEM has been shown to give reproducible results when multiple testing centers were compared.71 In trauma patients, ROTEM analysis has aided in early diagnosis of coagulation abnormalities.12 The prognostic value of ROTEM use in the emergency room has been reported, and CFT was found to be an independent predictor of death72 and to have a good negative predictive value with normal ROTEM values

unlikely to be associated with hemostatic disorders.73 ROTEM analysis has been used to assess bleeding risk in patients receiving cardiopulmonary bypass.74 Outside the surgical setting, ROTEM analysis has been used to assess the coagulation status in people with type II diabetes.75 In a study of cancer patients, there was signicant correlation between standard laboratory values and ROTEM results.76 The technology has also been used to monitor brinogen concentrate therapy in brinogen deciency77 and to evaluate neonatal hemostasis.78 ROTEM results were useful in predicting outcome in a prospective cohort study in septic patients79 and as a guide to blood transfusion approaches in cardiac patients.74 The ROTEM is being used to evaluate the capacity of LMWH to inhibit clot formation in patients undergoing angioplasty and stenting for carotid artery disease.80 Recently, PlateletMapping developed for the TEG has been validated for use on the ROTEM utilizing a slightly different concentration of the reagents.43 Application: veterinary medicine ROTEM analysis has been validated in horses with reference intervals established using citrated blood samples.81 Our laboratory has validated the ROTEM for dogs using citrated and native blood using the INTEM and EXTEM reagents. We are in the process of further applying this technology to other species and diseased populations (Figure 5).35,36,82 Advantages and limitations The use of a ball bearing system for power transduction is thought to make the ROTEM less susceptible to movement and vibration artifact. The presence of 4 channels allows up to 4 samples (or 2 with duplicates) to be run simultaneously. The ROTEM was designed to be sturdy and easily transportable for bedside monitoring. The electronic pipette can help to decrease

Figure 5. Abnormal ROTEM tracings. (A) A hypocoagulable state is indicated by prolonged clot time (CT) and clot formation time (CFT), decreased a, and decreased maximum clot rmness (MCF). (B) A hypercoagulable state is indicated by shortened CT and CFT, increased a, and increased MCF. Refer to Figure 3 for explanations of tracings and abbreviations.

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interoperator variability and makes the unit easier for nonlaboratory personnel to use. The ROTEM is more costly than the TEG and Sonoclot. Results from the 2 TE instruments, the TEG and ROTEM, cannot be compared directly.

Viscoelastic Testing
Advantages Use of viscoelastic POC instruments offers several advantages, including evaluation of whole blood and, therefore, the contribution of cells, rapid turn-around times as most pertinent information is available within 30 minutes, and small sample volumes, usually o 0.5 mL. Viscoelastic kinetic monitoring has the additional advantage of detecting hypercoagulability. Normal brinolysis is slow and not seen on typical tracings observed for up to 1 hour, but accelerated brinolysis can be identied on all 3 analyzers. Perhaps the greatest strength of this technology is its capacity to provide a global picture of the hemostatic process as coagulation is extremely complex in vivo. The summation picture provides an overall evaluation of all the components, including the cellular ones, of coagulation and is likely more clinically relevant than isolated plasma-based testing. Limitations The recent introduction of viscoelastic coagulation technology to veterinary medicine has resulted in signicant clinical use and multiple reports describing results obtained in several clinical populations. In many cases this technology is being reported as if it is a single diagnostic test with specic quantitative connotations. Unfortunately, there has been little recognition that viscoelastic monitoring, in particular with TEG, is a means of acquiring data about coagulation, rather than an individual diagnostic assay. The characteristics of the sample analyzed along with other additives have an enormous impact on interpretation of results. Reporting TEG results without considering sample characteristics, use of activators of coagulation, and sample-handling procedures that may have an impact on the results would be comparable to reporting coagulation tests without delineating whether the test performed was a PT, aPTT, or thrombin time. Different tests and test conditions yield vastly different information about the coagulation system.82 TE in general is not sensitive to mild coagulation factor deciencies or to mild defects of primary hemostasis.80 In several investigations, little to no effect of aspirin on TEG variables has been reported, even in the

presence of signicantly prolonged bleeding times.83,84 Defects in primary hemostasis, such as those resulting from treatment with aspirin or clopidogrel, can be detected with the PlateletMapping assay. Variations in HCT are known to affect the results of TE with increased and decreased HCT leading to tracings that indicate hypocoagulable and hypercoagulable states, respectively.35,36,40,8587 These effects have been reported in both in vitro dilution experiments and in animals with anemia or erythrocytosis. Whether this is truly a reection of in vivo effects of RBC mass on coagulation, or simply an artifact of TE technology, is not known at this time. Whole blood coagulation testing requires that a dened volume of whole blood be dispensed into the instrument; wide variations in HCT consequently result in wide variations in the volume of plasma loaded into the cup, which in turn has a marked impact on the total amount of coagulation proteins evaluated in the assay. Thus, HCT may be an important confounder in interpretation of viscoelastic test results. Note that in many studies results from diseased, and possibly anemic, animals are compared with references intervals established from animals with normal HCT. Dogs with IMHA, cancer, and chronic disease are often anemic; these populations are also reported to be in hypercoagulable states using TEG technology. These POC tests were designed to be performed within minutes of collection on fresh, whole, noncitrated blood. POC tests often lack stringent quality control that is applied to equipment in the clinical pathology laboratory; this may be one reason that they have been slow to gain favor clinically. Methods for performing TE have not been standardized by the Clinical and Laboratory Standard Institute, and this has hampered widespread acceptance. In a recent systematic review of TE studies, it was concluded that of 10 prospective TE studies eligible to be included in analysis only 5 reported measures of TEG accuracy.88 The overall quality of the studies was highly variable as were variations in the methodology, reference standards, and denitions of hypercoagulability; metaanalysis was not possible due to the wide variability in reporting. Comparison with standard plasma-based coagulation testing Although a direct comparison between viscoelastic and standard plasma-based test results is not possible, several correlations have been reported.57,89 For TEG, a signicant correlation between MA and brinogen concentration and platelet count has been reported in both normal and hypercoagulable people and dogs.37,51,64 Our laboratory recently reported

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correlation between plasma-based tests and comparable ROTEM values for normal dogs.82 Methods In classic TE, activators were not used and clotting was initiated by contact of the plasma component of the blood with the steel cup. The charge of the metal was a contact activator and resulted in production of FXIIa and subsequent downstream enzymes. With the introduction of plastic disposable cups, this activation became less effective and was more likely to be representative of a combination of contact activation, platelet activation, which can vary considerably depending on the level of preactivation of the sample, and the low level of tissue factor that is present in blood.45 In addition, performing all tests immediately was not practical in many situations. Anticoagulation with citrate had the advantage of better stability compared with nonanticoagulated blood; however, use of citrate has an impact on the results.44,90 Investigators have reported tracings indicative of hypercoagulability from citrated and recalcied samples compared with tracings of noncitrated samples.44 In addition to calcium, citrate chelates other metallic ions, such as magnesium and zinc, which are not added back upon recalcication and may have an important impact on coagulation. Furthermore, if citrated blood is recalcied in the absence of any activator, the delay between sample collection and recalcication has a profound impact on the results.82 This phenomenon occurs because activation of FXII is not calcium-dependent, and is not prevented by chelation of calcium by citrate.45 The nal TE tracing obtained is a function of FXIIa activity in the sample at the time of recalcication, which is dependent on FXIIa generated by surfaces, including the needle, syringe, and tube, to which blood was exposed and the inhibition of FXIIa by plasma inhibitors, primarily C1 inhibitor, in the sample. Consequently, use of citrated blood with recalcication without an activator increases the variability of the results obtained. Different types and concentrations of activators can be used with each of these instruments. Depending on the target of activation, ie, contact or tissue factor activation, the specic activator, eg, celite, kaolin, ellagic acid, and thromboplastin, and the concentration of activator used, test results will vary considerably.91 Contact activation can be achieved using kaolin, celite, or ellagic acid. Kaolin is a more potent activator of contact pathway than celite,92 and use of kaolin with both instruments has been reported.92 The quality of kaolin, a clay mineral that activates the con-

tact pathway, differs among mining sites and depends on impurities caused by the presence of other clays, with wide variations between manufacturers.92 The TEG-supplied reagent uses celite, whereas the ROTEM-supplied reagent uses ellagic acid. Relipidated tissue factor or thromboplastin is used for activation of the extrinsic pathway. The ROTEM reagents include a tissue factor reagent (EXTEM) that may aid in consistency of results. Reports with TEG describe dilution of commercially available PT reagents, primarily Innovin (Dade Behring, Marburg, Germany). A wide variety of dilutions are reported.41,58,64,70 Our laboratory recently compared several reagents used for initiating coagulation during TE, revealing that the potency and type of activator used strongly inuence results.82 The most profound effect was found on CT, which was expected as CT reects the time to initial brin polymerization and is primarily a function of the rate of initial thrombin formation. Latter phases rely more on contributions of platelets and brin polymerization than on initial thrombin burst, and, thus, the type of activator had less of an impact on CFT and a. The impact on MCF was minimal.82 Temperature is an important variable in all coagulation tests. Measurements should be made at 371C, and the cup and pin are both maintained at 371C. Cold cups, pins, or blood samples can slow reaction time producing falsely prolonged values. Standardization of sample warming procedures is essential for consistency. Operator variability is another factor and it is essential to establish instrument-specic and, in some cases, operator-specic reference intervals using standardized activators for each species.58

Conclusions
Viscoelastic testing with the Sonoclot, TEG, and ROTEM instruments is a new twist on an old technology and has the potential to markedly improve evaluation and management of hemostatic abnormalities. Ideal procedures for applying this technology and the patient populations that could benet most from viscoelastic testing are areas of active investigation in both human and veterinary medicine, with many issues remaining to be denitively resolved. For viscoelastic testing to be of optimal benet, standard protocols must be in place with respect to blood collection, including method of venipuncture, vacutainer tubes used, and order of lling tubes; temperature, with the sample maintained at 371C at all times or maintained at room temperature and then warmed to 371C; exact time from sample collection to assay;

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activator type and concentration; and other laboratory values, such as HCT.

follows orthotopic liver transplantation. Hepatology. 1990;12:553558. 11. Coakley M, Reddy K, Mackie I, Mallett S. Transfusion triggers in orthotopic liver transplantation: a comparison of the thromboelastometry analyzer, the thromboelastogram, and conventional coagulation tests. J Cardiothorac Vasc Anesth. 2006;20:548553. 12. Rugeri L, Levrat A, David JS, et al. Diagnosis of early coagulation abnormalities in trauma patients by rotation thrombelastography. J Thromb Haemost. 2007;5:289295. 13. von Kaulla KN, Ostendorf P, von KE. The impedance machine: a new bedside coagulation recording device. J Med. 1975;6:7388. 14. Hett DA, Walker D, Pilkington SN, Smith DC. Sonoclot analysis. Br J Anaesth. 1995;75:771776. 15. Dallap Schaer BL, Bentz AI, Boston RC, Palmer JE, Wilkins PA. Comparison of viscoelastic coagulation analysis and standard coagulation proles in critically ill neonatal foals to outcome. J Vet Emerg Crit Care. 2009;19:8895. 16. Dalbert S, Ganter MT, Furrer L, Klaghofer R, Zollinger A, Hofer CK. Effects of heparin, haemodilution and aprotinin on kaolin-based activated clotting time: in vitro comparison of two different point of care devices. Acta Anaesthesiol Scand. 2006;50:461468. 17. Tuman KJ, Spiess BD, McCarthy RJ, Ivankovich AD. Comparison of viscoelastic measures of coagulation after cardiopulmonary bypass. Anesth Analg. 1989;69:6975. 18. Pivalizza EG, Abramson DC, Harvey A. Perioperative hypercoagulability in uremic patients: a viscoelastic study. J Clin Anesth. 1997;9:442445. 19. Mukubo Y, Kawamata M. Perioperative hypercoagulability in patients with rheumatoid arthritis: Sonoclot study. J Anesth. 2004;18:6264. 20. Nilsson CU, Engstrom M. Monitoring fondaparinux with the Sonoclot. Blood Coagul Fibrinolysis. 2007; 18:619622. 21. Dallap Schaer BL, Wilkins PA, Boston R, Palmer J. Preliminary evaluation of hemostasis in neonatal foals using a viscoelastic coagulation and platelet function analyzer. J Vet Emerg Crit Care. 2009;19:8187. 22. Horlocker TT, Schroeder DR. Effect of age, gender, and platelet count on Sonoclot coagulation analysis in patients undergoing orthopedic operations. Mayo Clin Proc. 1997;72:214219. 23. Ekback G, Carlsson O, Schott U. Sonoclot coagulation analysis: a study of test variability. J Cardiothorac Vasc Anesth. 1999;13:393397.

Acknowledgments
The authors thank Elizabeth Rozanski, DVM, DACVIM, DACVECC, Associate Professor, Cummings School of Veterinary Medicine at Tufts University, for supplying the TEG tracings. Disclosure: The authors have indicated that they have no afliations or nancial involvement with any organization or entity with a nancial interest in, or in nancial competition with, the subject matter or materials discussed in this article.

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