Annemarie T. Kristensen, DVM, PhD, DACVIM, DECVIM (CA & ONC); Bo Wiinberg, DVM, PhD
The ability of a laboratory assay to reveal and correlate to clinical phenotype is crucial for rational hemostasis monitoring. The ideal hemostasis assay should therefore be able to identify both biochemical and cellular abnormalities in the hemostasis system and at the same time correlate to the clinical signs of the patient. The introduction of the cell based model of hemostasis, has made it evident that hemostasis is influenced by numerous pro- and anticoagulant components other than those present in blood plasma alone. It is important that the methods used for the diagnosis and monitoring of patients abnormal hemostasis take this into consideration. In particular TF expression in tissue and cellular components of the blood such as the activated platelets and leukocytes, supply a surface for initiation, amplification and propagation of clot formation and thus play a key role in hemostasis. These cellular and tissue components are themselves influenced by altered systemic inflammatory- and immune responses during disease.
The cell-based model of hemostasis has increased the understanding of the complex biochemistry of physiologic and pathologic hemostasis and has forced a re-evaluation of the traditional view of the intrinsic and extrinsic pathways of coagulation. With the knowledge that, whole blood contains all the intravascular factors and cells participating in physiologic and pathologic hemostasis, incorporating TF and phospholipid-bearing cells, it is reasonable to assume that whole blood assays such as thromboelastography (TEG) may provide a more accurate reflection of in vivo hemostasis than the traditionally used plasma based hemostasis assays.
TEG is not a new method, but its potential use in assessing hemostatic disorders has resurfaced after the assay was automated and new activators were introduced, allowing for rapid and global assessment of hemostatic function in whole blood.1 More specifically, TEG evaluates all of the steps in hemostasis, including initiation, amplification, and propagation as well as fibrinolysis, including the interaction of platelets and leukocytes with the proteins of the coagulation cascade. Thus, TEG combines evaluation of the traditional plasma components of coagulation with the cellular components.2 TEG has been used to evaluate hypercoagulability in dogs with parvoviral infection, and to evaluate platelet dysfunction in dogs with hypothermia.3,4 TEG has also been cited in a few abstracts, but the total amount of published material on dogs is sparse.5-9
Theoretically, all of the TEG parameters are influenced by abnormal hemostasis; R and K values are increased and α and MA values are decreased in hypocoagulable states and opposite changes are observed in hypercoagulable states. Thus, TEG analysis should be able to distinguish pathologic from physiologic states. The monitoring of hypocoagulable, and especially hypercoagulable, canine patients is difficult, both with regard to progression of disease and monitoring blood component and/or anticoagulation therapy.10-13 Thus TEG offers a welcome opportunity for patient near evaluation of overall haemostatic capability and could potentially provide clinicians with the ability to diagnose, monitor and predict therapeutic response in dogs with bleeding and/or thrombotic disorders. TEG analysis has the potential to aid in the diagnostic workup of patients with abnormal hemostasis and supplement the information received from traditional coagulation assays, such as PT, aPTT, D-dimer, and fibrinogen assays. Consequently TEG could have a major impact on how management of such patients is approached in the future and potentially help advance the treatment of these dogs.
Recent TEG Research in Dogs
Validation of TF-Activated TEG14
In this study TF activated TEG in dogs was validated, thus facilitating the use of the assay in future clinical research. The study demonstrated that canine citrated WB can be used for TEG analysis with human recombinant TF as the activator when stored at room temperature for T30 or T120. At both time points, a narrow range of measurements and low analytical variations were observed, suggesting TEG analysis may be of value in evaluating dogs with hemostatic disorders. A statistically significant difference was found between measurements on blood left at room temperature for 30 versus 120 min, with at clear trend towards hypercoagulability with time. This trend towards hypercoagulability can be explained by the fact that citrated blood does not completely inhibit thrombin formation or the subsequent activation of coagulation.15 The difference between the 30 and 120 minute measurements indicated that, when using serial TEG measurements to monitor a patient, a fixed time point after sampling should be chosen to avoid the risk of inter-assay variation in TEG measurements exceeding the actual physiologic or pathologic hemostatic changes in the patient.
Correlation to Clinical Signs of Bleeding16
This study demonstrated that TF activated TEG was able to correctly identify dogs with clinical signs of bleeding with both higher positive- (PPV) and negative predictive values (NPV) than the conventional coagulation profiles which are widely used in veterinary medicine. The results further indicated that, TEG G is easier for the clinician to interpret than the combination of tests comprising the coagulation profile used in this study. The results also showed that TEG can be used to differentiate between dogs that are hypo- normo- and hypercoagulable.
In addition the results suggested that TF-activated TEG can be used as a screening tool to assess the overall haemostatic state of patients suspected of being at risk of bleeding due to haemostatic dysfunction, and that it correlates better and in a simpler and objectively interpretable manner to clinical symptoms, than the traditionally used coagulation screen. This should also allow monitoring of therapeutic measures.
The findings observed in the study substantiate, that an assay including cellular as well as plasma components may give a more reliable evaluation of the overall status of the haemostatic capability of the patient. With the apparent ability to detect hypercoagulability by thromboelastography, the potential for prospective evaluation of correlation of hypercoagulability to thrombosis should be possible.
In a study examining the application of TF-TEG as an aid in the diagnosis of DIC, it was demonstrated that TEG can be used to distinguish between different stages of DIC in dogs. The most common overall hemostatic abnormality in this group of dogs was hypercoagulability and interestingly the results showed that there was a significant 2.4x higher relative risk of 28 day mortality in dogs that were hypocoagulable on TEG compared to those that were hypercoagulable, demonstrating the potential use of the assay as a prognostic indicator. The finding that mortality was significantly lower in the hypercoagulable group, supports the assumption from humans, that early and aggressive intervention at the stage of non-overt DIC, if identifiable, may be vital for outcome also in dogs. With the ability to detect hypercoagulability in DIC, TEG provides the clinician with the ability to identify dogs that are in the early pro-inflammatory and hypercoagulable state of DIC and offers the novel possibility of clearly differentiating this group of dogs from those in the later consumptive but still non-overt stages of DIC and as such, TEG as a test of global hemostasis may potentially provide us with an option for more individually tailored treatment plans for patients with DIC in the near future, which could have a positive effect on the ability to treat these dogs with DIC and the resulting outcome.
Cancer is the most common disease related cause of death in dogs. Cancer has been known to cause hemostatic abnormalities in up to 80% of canine cancer patients, believed to result in bleeding complications and an association between venous thromboembolic disease and cancer has also been observed. In a recent study, the overall hemostatic functional state including hypercoagulability was assessed using citrated whole blood. In the 36 dogs with malignant neoplasia 18/36 (50%) were hypercoagulable, whereas 6/36 (17%) were hypocoagulable. All hypocoagulable dogs had metastatic disease. The proportion of dogs with altered hemostasis was significantly different between dogs with malignant and benign neoplasia and it was documented for the first time that the most common abnormality is hypercoagulability, similar to what is observed in humans, allowing us to use TEG to identify and potentially treat these patients. In addition, these findings indicate that dogs with malignant neoplasia and hypercoagulability may serve as a model for human disease. A follow up study has confirmed that hypercoagulability is the most common hemostatic abnormality in canine cancer patients whether of hemaotopoietic, epithelial or mesenchymal origin. Interestingly TEG results in dogs with metastatic disease varied according to tissue type. In dogs with metastatic carcinoma, 60% were hypocoagulable. Additional studies are needed to show whether therapy antagonizing one of the components in hemostasis is of benefit in these dogs and whether or not the TEG assay will be of value in monitoring patients receiving such therapy.
A Therapeutic Guide?
In humans, TEG is increasingly used to monitor hemostatic function in cardiac and hepatic surgery and to optimize blood-product selection and usage, but the role of TEG is now expanding to also include platelet mapping assays, as well as diagnosis and treatment of both hypocoagulable- and hypercoagulable states.17-21 Especially the use of TEG as a transfusion guide seems to have a large potential in veterinary medicine, as this is intuitively easy perform and to interpret. Furthermore it has the potential to significantly optimize blood product usage and efficacy.
But TEG also has the potential to test the efficacy of different types of medication in vitro. An example of this is an in vitro study, where we endeavored to evaluate heparinase modified TEG as a possible method to evaluate the effect of therapy with the low molecular weight heparin (LMWH) dalteparin.(In press VCP) The results showed that spiking citrated canine whole blood with increasing doses of dalteparin significantly and dose-dependently affected all TF-activated TEG parameters. In contrast to this, it was observed that when using Kaolin as an activator there was almost no measurable dalteparin effect, with statistically significant dose-dependent alterations seen only for the R value.
The result of this study indicate that when coagulation is triggered by diluted tissue factor, only some of the anti-coagulant mechanisms of dalteparin are reversed by heparinase. The exact mechanisms responsible for this are not known, but one possible explanation could involve mechanisms related to the function of TFPI, which exerts its anticoagulant action by forming a quaternary complex with the FXa-TF-FVIIa complex. The results of this study gives reasonable evidence that the TF activated TEG assay may help provide information on the global haemostatic activity of LMWH. The results of this study show that increasing concentrations of dalteparin pushes the TEG tracing to the right and narrows the height of the tracing in a dose dependent manner. This unique feature of the TEG assay suggests that it could be utilized in patient near targeting of therapy of the hypercoagulable patient towards a normalization of the patients TEG tracings and hereby tailor the LMWH dose to meet the requirement of the individual patient, something not possible with currently used methods. Prospective clinical studies are required to evaluate the clinical usefulness of heparinase modified TEG in monitoring of anticoagulant therapy with LMWH.
The results so far in canine studies of tissue factor activated-TEG appear promising in the diagnostic phase, allowing identification of the overall hemostatic state of the patient as hypo-, normo-, or hypercoagulable. In addition, it appears to enable an individualized patient near approach to guiding blood component therapy, anti-and procoagulant therapy as well as a tool for monitoring the patient continuously during therapy.
1. Hartert H. Blutgerinnungsstudien Mit der Thrombelastographie, Einem Neuen Untersuchungsverfahren. Klinische Wochenschrift 1948;26:577-583.
2. Mallett SV, Cox DJ. Thrombelastography. Br J Anaesth 1992;69:307-313.
3. Otto CM, Rieser TM, Brooks MB, et al. Evidence of hypercoagulability in dogs with parvoviral enteritis. J Am Vet Med Assoc 2000;217:1500-1504.
4. Ao H, Moon JK, Tashiro M, et al. Delayed platelet dysfunction in prolonged induced canine hypothermia. Resuscitation 2001;51:83-90.
5. Hoffman KN, Traverso CI, Arcelus JI, et al. Thromboelastography Versus Other Hemostatic Tests for Monitoring Experimental Cardiopulmonary Bypass on Dogs [abstract]. Thromb Haemost. 1993;69:2244.
6. Tuman KJ, Mccarthy RJ, Patel RB, Ivankovich AD. Quantification of Aprotinin Reversal of Severe Fibrinolysis in Dogs Using Thromboelastography [abstract]. Anesthesia and Analgesia. 1993;76:S439.
7. Mousa S. Synergistic interactioons between GPIIb/IIIa antagonists and low molecular weight heparin in inhibiting platelet-fibrin clot dynamics in human blood and in canine model using thromboelastography [abstract]. Blood. 2002;100:3986.
8. Tuman K, Naylor B, Spiess B, McCarthy R, Ivankovich A. Effects of hematocrit on thromboelastography and sonoclot analysis [abstract]. Anesthesiology. 1989;71:A414.
9. Spiess B, McCarthy R, Ivankovich A. Primary fibrinolysis or D.I.C. differentiated by different viscoelastic tests [abstract]. Anesthesiology. 1989;71:A415.
10. Ross S, Smith S, Lekcharoensuk C. Disseminated intravascular coagulation (DIC) in dogs: 252 cases (1999-2000) [abstract]. J Vet Intern Med. 2002;16:350.
11. Logan JC, Callan MB, Drew K, et al. Clinical indications for use of fresh frozen plasma in dogs: 74 dogs (October through December 1999). J Am Vet Med Assoc 2001;218:1449-1455.
12. Lindblad B, Borgstrom A, Wakefield TW et al. Haemodynamic and haematologic alterations with protamine reversal of anticoagulation: comparison of standard heparin and a low molecular weight heparin fragment. Eur J Vasc Surg 1987;1:181-185.
13. Palmer KG, King LG, Van Winkle TJ. Clinical manifestations and associated disease syndromes in dogs with cranial vena cava thrombosis: 17 cases (1989-1996). J Am Vet Med Assoc 1998;213:220-224.
14. Wiinberg B, Jensen AL, Rojkjaer R, et al. Validation of human recombinant tissue factor-activated thromboelastography on citrated whole blood from clinically healthy dogs. Vet Clin Pathol 2005;34:389-393.
15. Camenzind V, Bombeli T, Seifert B, et al. Citrate storage affects Thrombelastograph analysis. Anesthesiology 2000;92:1242-1249.
16. Wiinberg B, Jensen AL, Rozanski E, et al. Tissue factor activated thromboelastography correlates to clinical signs of bleeding in dogs. Vet J 2007;
17. Kang YG, Martin DJ, Marquez J, et al. Intraoperative changes in blood coagulation and thrombelastographic monitoring in liver transplantation. Anesth Analg 1985;64:888-896.
18. Spiess BD, Tuman KJ, Mccarthy RJ, et al. Thromboelastography as an indicator of post-cardiopulmonary bypass coagulopathies. J Clin Monit 1987;3:25-30.
19. Thomson A, Napier JA, Wood JK. Use and abuse of fresh frozen plasma. Br J Anaesth 1992;68:237-238.
20. Gonano C, Sitzwohl C, Meitner E et al. Four-day antithrombin therapy does not seem to attenuate hypercoagulability in patients suffering from sepsis. Crit Care 2006;10:R160.
21. Ben-Ari Z, Panagou M, Patch D, et al. Hypercoagulability in patients with primary biliary cirrhosis and primary sclerosing cholangitis evaluated by thrombelastography. J Hepatol 1997;26:554-559.