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Keywords:

  • Assay;
  • Canine;
  • Hemostasis;
  • Plasma;
  • TEG

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

Background: There is considerable variation in the coagulation profile of dogs with disseminated intravascular coagulation (DIC), making it difficult to assess overall hemostatic function.

Objectives: To characterize the overall hemostatic state in dogs with DIC, by use of tissue factor-activated thromboelastography (TF-TEG), and to determine whether there is an association between hemostasis and outcome.

Animals: 50 dogs with DIC.

Methods: Dogs admitted to the intensive care units, with an underlying disease known to predispose to DIC, were prospectively assessed with TF-TEG. Citrated blood samples were collected daily during hospitalization and an extended coagulation panel and TF-TEG were performed. Diagnosis of DIC was based on expert opinion.

Results: Hemostatic dysfunction was observed on the TF-TEG profile in 33/50 of the dogs, of which 22/50 were hypercoagulable and 11/50 were hypocoagulable based on the TF-TEG G value alone. There were significant differences in k, α, and MA values (P < .0001) among hypo-, normo-, and hypercoagulable dogs. There was a significant difference in case fatality rate between hypo- (64%) and hypercoagulable (32%) dogs (relative risk = 2.38; P= .04). Dogs that died had significantly lower antithrombin activity (P= .03) and higher d-dimer concentration (P= .03) than survivors.

Conclusions: The most common overall hemostatic abnormality in dogs diagnosed with DIC was hypercoagulability, and there was significant difference in survival between hyper- and hypocoagulable dogs. The results suggest TF-TEG is valuable in the assessment of hemostatic function in dogs diagnosed with DIC.

Disseminated intravascular coagulation (DIC) is a complicated and dynamic hemostatic disorder characterized by variable imbalances of the components of the hemostatic system. DIC always occurs secondary to an underlying disease that, among others, causes an uncontrolled systemic inflammatory response.1,2 In the initial stage of DIC, the patient is thought to be hypercoagulable because of circulating inflammatory mediators, which cause activation of hemostasis through increased exposure of tissue factor (TF) as well as inhibitor consumption.2 Consumption of coagulation factors and increased fibrinolytic activity, if not compensated, can lead to a hypocoagulable state with overt clinical symptoms of bleeding.3 Owing to the progressive nature of DIC, the clinical signs vary considerably and range from no signs of DIC (nonovert DIC), accompanied by no or mild changes in traditional hemostasis parameters (activated partial thromboplastin time [aPTT], prothrombin time [PT], d-dimer, fibrinogen, and platelet count), to signs of organ failure, associated with microvascular thrombosis in vital organs, finally culminating in overt bleeding symptoms (overt DIC).1,3 The diverse clinical characteristics of DIC make initial diagnosis and optimization of treatment very challenging.

The traditional theoretical and diagnostic approach to the hemostatic system divides it into primary hemostasis (vascular tone and the platelet plug), secondary hemostasis (coagulation), fibrinolysis (breakdown of the clot), and the presence of endogenous anticoagulants, which limit clot formation to the site of injury. Accordingly, assessment of a dog's hemostatic capabilities in DIC is traditionally performed with tests of primary hemostasis, including platelet count and platelet function tests, and secondary hemostasis through plasma-based assays designed to further localize defects such as the aPTT (intrinsic pathway) and PT (extrinsic pathway). The fibrinolytic system is traditionally evaluated with measurements of degradation products, including fibrinogen degradation products (FDPs) and d-dimer. Endogenous antithrombotic ability has been evaluated through measurement of antithrombin activity (AT) and concentrations of protein C (PC) and protein S (PS).4 Specialized individual coagulation factor tests can be performed to further localize the defect. All of these tests of the secondary and fibrinolytic systems are performed on citrated plasma samples and target very specific elements in the hemostatic system and thus potentially discount important cellular factors. Although this approach makes it possible to diagnose DIC effectively and systematically, it can be difficult from a clinical perspective to piece together a picture of a dog's overall hemostatic capability and to predict or monitor the effect of treatment with anticoagulant or procoagulant medication with this traditional approach, especially if the dog is suspected of being hypercoagulable.

The recent discovery of the cell-based, TF/factor VII-dependent model of hemostasis has increased our understanding of the complex biochemistry of physiologic hemostasis and has forced a re-evaluation of the traditional view of the intrinsic and extrinsic pathways of coagulation.5,6 Concurrently, the important role of activated platelets in amplification of thrombin generation in the cell-based hemostasis model has been recognized.7–9 In inflammation, cytokine-induced platelet activation and TF expression on cytokine-activated mononuclear cells lead to the systemic activation of coagulation.10–13 Although citrated plasma contains many of the factors involved in coagulation, whole blood contains both the soluble factors and intravascular cells active in physiologic and pathologic hemostasis, incorporating TF and phospholipid-bearing cells such as platelets and leukocytes. Although thromboelastography (TEG) is not a new method, 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 in dogs.14 More specifically, TEG evaluates all 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.15 Recently, a TF-activated TEG assay on citrated whole blood has been validated in dogs, and the assay has been shown to have a low analytical variation compared with many of the traditional plasma-based coagulation assays in normal healthy dogs.14,16 TEG has been used to evaluate hypercoagulability in dogs with parvoviral infection and to evaluate platelet dysfunction in dogs with hypothermia.17,18 TEG has also been cited in a few abstracts, but the total amount of published material on dogs is sparse.a,b,c,d TEG is increasingly used to monitor hemostatic function in humans after cardiac and hepatic surgery and to optimize blood-product selection and usage, but the role of TEG also includes platelet-mapping assays as well as diagnosis and treatment of both hypo- and hypercoagulable states.19–23

With DIC, the major challenge for the clinician is to make the diagnosis in the early, hypercoagulable, and nonovert stage of the disease. There is general consensus that early intervention in humans with DIC increases the patient's chances of survival. Although TEG cannot eliminate the need for plasma-based coagulation analyses in the initial diagnosis of DIC in dogs, it can be used to detect hypo-, normo-, and hypercoagulability, potentially allowing for more distinct stratification of dogs with DIC before treatment. Therefore, the objective of the present study was to characterize the overall hemostatic state in dogs presenting with DIC irrespective of clinical signs of bleeding, using the newly validated TEG assay, with the overall aims of (1) differentiating among hypo-, normo-, and hypercoagulable subgroups of DIC with TF-TEG and (2) examining whether there is an association between subgroup of DIC and outcome.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

This study was approved by the Small Animal Ethics and Administrative Committee at the Department of Small Animal Clinical Sciences, Faculty of Life Sciences, University of Copenhagen, Denmark and by the Institutional Animal Care and Use (IACUC) Committee at Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA. The study was performed as a prospective multicenter observational study, during a 2-year period from 2004 to 2006 at the Department of Small Animal Clinical Sciences, Faculty of Life Sciences, University of Copenhagen, Denmark and Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA.

Inclusion and Exclusion Criteria

Eligible dogs were those admitted to the intensive care unit (ICU) at one of the above-mentioned hospitals, with an underlying disease known to predispose to DIC (Table 1) and a clinical suspicion of DIC, where the primary clinician ordered a coagulation profile consisting of aPTT, PT, d-dimer, fibrinogen, and a platelet count. All eligible dogs during the study period were included consecutively and 150 dogs were evaluated for the presence of DIC. To eliminate the effect of multiple sampling on blood volume, only dogs weighing more than 8 kg were included in the study to ensure that <1% of total blood volume was collected daily. Sixty-three dogs were seen in Copenhagen over a 21-month period and 50 dogs in North Grafton over a 3-month period. Potential cases were included when the dogs were admitted to the emergency room (ER) or ICU and the 1st blood samples drawn before treatment was commenced. If samples were not collected at the time of admission or if time points were missed, the dogs were subsequently excluded from the study. The dogs were evaluated with a TF-activated TEG assay upon admission and then daily for the duration of their hospitalization in ICU. All specialized coagulation assays (AT, PC, PS, plasminogen [PLG], and α2-APL) were performed retrospectively at the central laboratory at the Faculty of Life Sciences, University of Copenhagen, Copenhagen, Denmark. Dogs treated with heparin, aspirin, or other anticoagulant therapy within 48 hours before sampling as well as dogs suffering from von Willebrand factor (vWF) deficiency, chronic heart disease, or chronic hepatic disease were excluded from the study.

Table 1.   Frequency of underlying diseases in 50 dogs diagnosed with disseminated intravascular coagulation.
List and Frequency of Clinical Diagnosis of 50 Dogs Diagnosed with DIC, Divided into Groups Based on Their Overall Hemostatic Capability Evaluated with TF-Activated TEG
  1. IMHA, immune mediated hemolytic anemia; IMTP, immune mediated thrombocytopenia.

Hypocoagulable (n = 11)
Hemangiosarcoma (3)
Mammary adenocarcinoma (2)
Splenic tumor (2)
Gastric dilatation volvulus
IMHA + IMTP
IMTP
Lymphoma
Normal (n = 17)
Multitrauma (3)
Lymphoma (2)
Sepsis (2)
Acute hepatitis
Angiostrongylus vasorum
Gastric dilatation volvulus
Heartbase tumor
Leukemia
Malignant histiocytosis
Pancreatitis
Pulmonary hemorrhage
Rattlesnake bite
Splenic tumor
Hypercoagulable (n = 22)
Angiostrongylus Vasorum (4)
IMHA (4)
Sepsis (4)
Acute hepatitis (2)
Pancreatitis (2)
Foreign body
Gastric dilatation volvulus
Hemorrhagic gastroenteritis
Intraabdominal sarcoma
Lymphoma
Mammary adenocarcinoma

Sampling

Blood samples were collected upon hospitalization and then once daily from the included dogs. Whole blood was collected by careful jugular venipuncture, using minimum stasis and a 21-G butterfly needle, or in dogs that had central venous lines, blood was drawn from these with syringes. The 1st 10 mL from the central venous line was then discarded to make sure that there was no heparin left in the catheter before blood collection. Blood samples were collected into 1 serum, 2 citrated, and 1 EDTA vacutainer plastic tubes,e in that order. The EDTA blood sample was used for platelet count. The 3-mL citrate tubes were inverted carefully 5 times after sampling, to ensure mixing of 3.2% trisodium citrate and blood in a 1 : 9 ratio, and either stored at room temperature for subsequent TEG analysis or centrifuged immediately at 4,000 × g for 120 seconds. The plasma was collected from the centrifuged tubes within 30 minutes of sampling and a coagulation profile was performed immediately or the plasma was stored at −80 °C until coagulation profile analysis was possible.

TEG

TEG analyses were carried out 30 minutes after blood sampling at both the Central Laboratory, Department of Small Animal Clinical Sciences, Faculty of Life Sciences, University of Copenhagen and at Tufts University. The clinicians were not blinded to the results of the analyses.

The TEG analyses were performed with the same model of a computerized thromboelastographf according to the previously published method.14,16 In brief, data were obtained continuously by electronic transfer to computer from the analyzer. Canine citrated whole blood samples were activated with a solution of recombinant human TFg prediluted 1 : 2,780 in a HEPES buffer with 2% bovine serum albumin (BSA). Two hundred and eighty millimolar CaCl2 was added to the cup before analysis giving a total volume of 360 μL/cup of reagents and canine citrated whole blood in each experiment and a final TF dilution of 1:50,000.

The TEG analyses were run for 120 minutes with measurement of global clot strength (G), reaction time (R), clotting time (K), angle (α), and maximum amplitude (MA). Based on the TEG G value, dogs were characterized as hypo- (<3,200), normo- (G= 3,200–7,200), and hypercoagulable (G > 7,200). The results of TEG measurements were compared with a previously established reference range.14,16 TEG global clot strength is referred to as G, where G= 5,000 × MA/(100 − MA), and is a measure of the overall coagulant state as normo-, hyper-, or hypocoagulant. The TEG values used in the present study are the reaction time (R), which is the time of latency, from the time blood is placed in the TEG analyzer until initial fibrin formation, measured as an increase in amplitude of 2 mm; it is primarily related to plasma clotting factors and inhibitor activity. The clotting time (k) is the time to clot formation, measured from the end of R until amplitude of 20 mm is reached; it is a measure of the time it takes from initial clot formation until predetermined clot strength is reached, and is primarily related to clotting factors, fibrinogen, and platelets. The angle (α) represents the rapidity of fibrin build-up and cross-linking, and is mainly dependent on the concentrations of platelets, fibrinogen, and clotting factors. The MA is a direct function of the fibrin and platelet bonding, which represents the ultimate strength of the fibrin clot. Theoretically, all the TEG parameters examined in this study 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 (Fig 1). Thus, TEG analysis should be able to distinguish most pathologic from physiologic states and may potentially give a more complete picture of the patients in vivo hemostatic capabilities.

image

Figure 1.  Pattern recognition chart of TEG tracings pertaining to disseminated intravascular coagulation, compiled from human research. R, reaction time; K, clotting time; α, angle; MA, maximum amplitude. TEG, thromboelastography.

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Coagulation Profile

Platelet count was performed locally at the time of sampling. All other hemostasis tests used for the diagnosis of DIC were performed at the Central Laboratory, Department of Small Animal Clinical Sciences, Faculty of Life Sciences, University of Copenhagen. The coagulation profile included aPTT, PT, fibrinogen, d-dimer, PC, PS, α2-antiplasmin, and PLG. aPTT, PT, PC, PS, α2-antiplasmin, PLG, and fibrinogen were measured with an automated hemostasis analyzer,h platelet concentration with an automated CBC instrument,i and concentrations of d-dimer with an immunometric flow-through principle.j A list of reagents used is provided in Table 2. Plasma samples were thawed at 37 °C in a water bath immediately before analysis and centrifuged at 4,000 ×g for 3 minutes (to avoid remnants of cryoprecipitate in plasma after thawing); the supernatants were used for analysis. A pooled sample of plasma from 5 clinically healthy dogs was analyzed together with the samples and used as an estimated reference value. Results of the coagulation assays were defined as abnormal if they deviated from the reference pool values by more than 20%.

Table 2.   Laboratory coagulation assays run on automated hemostasis analyzer ACL 9000.
FactorsMethodsUnitsKits, Instrumentation Laboratory, USA
  1. aPTT, activated partial thromboplastin time; PT, prothrombin time; Fib, fibrinogen; AT, antithrombin; PC, protein C; PS, protein S; PLG, plasminogen; αPLI, α2antiplasmin inhibitor.

aPTTClotting timesecondsAPTT-SP (liquid) (0020006300)
PTClotting timesecondsPT-fibrinogen recombinant (20005000)
FibClotting timeg/LPT-fibrinogen (9756710)
ATChromogenic substrate%Liquid antithrombin (0020002500)
PCChromogenic substrate%Protein C (0020009100)
PSChromogenic substrate%Protein S (0020002800)
PLGChromogenic substrate%Plasminogen (0020009000)
αPLIChromogenic substrate%Plasmin inhibitor (0020009200)

Diagnosis of DIC

Owing to the lack of a gold standard, an approach similar to that used by Bakhtiari et al.24 was adopted for use in this study. This approach uses an expert panel as gold standard for diagnosis of DIC. The experts are given the results of a wide range of hemostasis assays and asked to identify patients with DIC based on the criteria defined by R.L. Bick, namely that a patient suffering from DIC should have evidence of procoagulant activation, inhibitor consumption, and increased fibrinolytic activity.3

To this end, an expert panel composed of 1 human physician and 2 veterinarians, all with >10 years experience in working with patients with inflammatory and hemostatic abnormalities, was used. Diagnosis was derived from blinded expert evaluation of the results of the extended coagulation profile (aPTT, PT, d-dimer, fibrinogen, PC, PS, AT, PLG, α2-antiplasmin, and a platelet count). To limit bias, the experts were blinded to the results of the other experts (test review bias) and clinical information about the patients other than underlying disease (clinical review bias).25,26 To establish the final diagnosis of DIC, the experts were asked to identify abnormalities in the coagulation profile fulfilling the criteria for the human approach to DIC defined by The International Society for Thrombosis and Hemostasis (ISTH), eg, activation of coagulation (PT, aPTT, and platelet count), inhibitor consumption (AT, PC, and PS), and increased fibrinolytic activity (d-dimer, PLG, and α2-antiplasmin).1,3 The final diagnosis was based on simple majority.

Statistical Analysis

Distribution of the data was assessed by the D'Agostino and Pearson omnibus normality test. Because values were not normally distributed, the statistical analyses were carried out as nonparametric. The TEG data from the day with the highest DIC score were used for statistical analysis. A χ2-test was used to assess whether there was a significant difference in 28-day mortality among hypo-, normo-, and hypercoagulable dogs based on TEG G value. Relative risk (RR) for 28-day mortality irrespective of cause, including euthanasia, and for TEG G was calculated based on the cutoff values for hypo- (<3,200 dyn/cm2), normo- (3,200–7,200 dyn/cm2), and hypercoagulable (>7,200 dyn/cm2) results. For the coagulation assays, RR was calculated based on a cutoff value of d-dimer <0.5 mg/L and AT activity <80%. A Spearman-ranked correlation analysis was applied to identify correlations between TEG G value and components of the coagulation profile. A Mann-Whitney U-test was used to assess whether there was any difference in TEG G or the individual components of the coagulation profile between survivors and nonsurvivors. A Kruskal-Wallis ANOVA, with a Dunn's multiple comparisons posttest, was applied, with the null hypothesis that there were no differences among the R, K, α, and MA values among hypo-, normo, and hypercoagulable groups. Statistical significance was set at P < .05. Statistical softwarek was used for all statistical analyses.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

Animals

Fifty dogs with DIC were detected during the study period. Twenty-three dogs were seen in Copenhagen and 27 in North Grafton. Table 1 shows the prevalence of underlying disease in the 50 included dogs. There were 13 intact males (median age 4.4 years), 10 castrated males (median age 9.1 years), 13 intact females (median age 6.0 years), and 14 spayed females (median age 6.0. years) in the group. Eight of the dogs were Labrador Retrievers, 4 were mixed breeds, 3 were Dachshunds, and all other breeds were represented only once or twice. For all dogs, clinical signs of bleeding were noted at the time of analysis. The dogs were considered to have clinical signs of a bleeding disorder if they had muco-cutaneous bleeding such as petecchia and ecchymoses, hematoma formation, hemothorax, hemoperitoneum, hemopericardium, or hemarthrosis. They were not considered to have clinical signs of a systemic bleeding disorder if they had local bleeding from the gastrointestinal tract because of hemorrhagic gastroenteritis, hematuria because of cystitis, or a small local bleeding such as a localized skin wound. One dog had a missing aPTT value and 3 dogs had fibrinogen levels over the measurable range of the assay.

TEG

There were 11 (22%) hypo- (TEG G <3,200), 17 (34%) normo- (TEG G 3,200–7,200), and 22 (44%) hypercoagulable (TEG G > 7,200) dogs based on TEG G value. Further, 9/11 (82%) hypo-, 2/17 (12%) normo-, and 1/22 (5%) hypercoagulable dogs had overt DIC with clinical symptoms of bleeding. Overall case fatality rate was 24/50 (48%). Case fatality rate was 7/11 (64%) for hypo-, 10/17 (59%) for normo-, and 7/22 (32%) for hypercoagulable dogs. When classified into hypo-, normo-, and hypercoagulable groups based on TEG G value, there was a statistically significant difference in outcome (28-day mortality) between hyper- and normocoagulable dogs (P < .05; RR = 1.85) and between hyper- and hypocoagulable dogs (P < .05; RR 2.38). The R, k, α, and MA TEG values for the hypo-, normo-, and hypercoagulable groups of dogs are shown in Figure 2.

image

Figure 2.  Results of the thromboelastograph (TEG) parameters R, reaction time; K, clotting time; α, angle; MA, maximum amplitude from 50 dogs diagnosed with disseminated intravascular coagulation. The results are divided into groups of hypo-, normo-, and hypercoagulable dogs based on TEG G value.

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Coagulation Profile

When comparing the results of the coagulation panel, stratifying for hypo-, normo-, or hypercoagulable TEG results (Table 3), there were statistically significant differences in platelet count (P= .0004), fibrinogen (P= .0004), and PLG (P= .0081). Dunn's multiple comparison indicated this difference was between hypo- and hypercoagulable groups (P < .001 for platelet count, P < .01 for fibrinogen, and P < .05 for PLG), and between hyper- and normocoagulable groups (P < .05 for platelet count, P < .01 for fibrinogen, and P < .05 for PLG). There was no significant difference between hypo- and normocoagulable groups. In the plasma-based coagulation assays there was a significant difference between survivors and nonsurvivors, with increased d-dimer (P= .0261) and decreased AT% (P= .0298) in dogs that died. Although there was a clear trend toward increased RR of mortality in dogs with low AT or high d-dimer, this difference was not statistically significant compared with those with normal values (Fig 3).

Table 3.   Median (range) of all of the coagulation analyses as they relate to the TEG stage (hyper, normal, and hypo).
 Hypocoagulable (n = 11)Normocoagulable (n = 17)Hypercoagulable (n = 22)
  1. *Significant difference between groups, P<.05.

Platelet count (109/L)*65 (6–200)146 (29–441)200 (54–507)
Fibrinogen (g/L)*1.6 (0.4–4.2)2.4 (0.2–6.7)6.1 (1.0–11.7)
PT (seconds)9.0 (7.8–43.7)8.1 (6.4–17.2)7.8 (6.5–10.2)
aPTT (seconds)14.2 (11.7–61.0)16.1 (11.2–110.0)16.7 (11.9–60.5)
d-dimer (mg/L)2.1 (0.3–5.7)0.9 (0.2–6.5)0.8 (0.2–3.5)
Antithrombin (%)69 (44–93)66 (19–128)68 (32–128)
Protein C (%)67 (28–90)90 (13–115)82 (48–157)
Protein S (%)75 (23–134)61 (5–135)88 (57–145)
Plasminogen (%)*58 (22–82)58 (10–195)89 (37–192)
α2-antiplasmin (%)78 (43–135)69 (42––130)88 (27–138)
image

Figure 3.  Graphs of significant correlations between thromboelastograph G value and different hemostasis assays from 50 dogs diagnosed with disseminated intravascular coagulation. The broken lines indicate upper and lower limits of guideline reference ranges.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

This study demonstrates that with TF-activated TEG, it is possible to distinguish between different stages of DIC in dogs and that the most common overall hemostatic abnormality in dogs with DIC is hypercoagulability. There was a 2.4 × higher RR of 28-day mortality in dogs that were hypocoagulable on TEG compared with those that were hypercoagulable, demonstrating the potential use of the assay as a prognostic indicator. The low AT level and high d-dimer in 28-day nonsurvivors compared with 28-day survivors suggest severe thromboembolic disease in this group of dogs; however, these markers were not able to demonstrate prognostic significance. This finding is in accordance with the findings of several previous studies.27–30

Laboratory testing in the management of any clinical condition is relevant only if it can be used to indicate and guide the appropriate institution of therapeutic measures or monitor the status of a disease state. Although useful in its indication of worsening in the primary condition or raising awareness of otherwise unsuspected disease, there has been no noticeable breakthrough as to how recognition of hemostatic dysfunction in dogs with DIC could affect outcome. As such, meaningful progress in DIC testing in dogs over the years has largely faltered, because there has been no universally proven diagnostic tests or therapy for DIC. A plausible explanation for the lacking progress in diagnosis and treatment of DIC is that the laboratory diagnosis of DIC in dogs is not standardized and therefore the hemostatic function tests used are not consistent. In veterinary medicine, DIC is often diagnosed based on three or more abnormal hemostatic parameters such as aPTT, PT, fibrinogen, d-dimer, platelet count, and red blood cell morphology, along with predisposing disease, which is a sensitive but unspecific approach. Currently, there is neither consensus nor an obvious golden standard for the diagnosis of DIC in dogs. In an attempt to increase both sensitivity and specificity of diagnosis of DIC, in this study diagnosis was based on expert evaluation of an extended coagulation panel. A similar approach has recently been used in a human study evaluating applicability of a scoring scheme developed by the ISTH for the diagnosis of DIC.24

This study also demonstrates that with TF-activated TEG, it is possible to differentiate among subgroups of DIC in dogs. Accompanied by the finding that case fatality rate was significantly lower in the hypercoagulable group, it supports the assumption that early or aggressive intervention in dogs with DIC might be associated with outcome. This is in accordance with interpretation from humans, where it is believed that aggressive intervention in the early hypercoagulable stage of DIC, through supportive or antithrombotic therapy, while the underlying disease is treated, may minimize thromboembolic complications and delay or even prevent progression to overt signs, thus increasing the individual's chances of survival.3,31–33 Assessment of hypercoagulability and thrombosis in dogs is very difficult with routinely used coagulation assays such as d-dimer, which has mainly negative predictive value for thromboembolism.34,35 Thus, there has been a need for improved assay methods that enable easy and near patient assessment of the overall hemostatic state. With the ability to detect hypercoagulability in DIC, TEG provides the clinician with the unique ability to identify dogs that are in the proinflammatory and hypercoagulable state of DIC and offers the novel possibility of clearly differentiating this group of dogs from those in a consumptive but still nonovert stage of DIC, and as such, TEG may potentially be useful for individualization of treatment. Further studies are needed to address whether hypercoagulable dogs with DIC will benefit from therapeutic intervention by anticoagulant therapy, and whether TEG can be used to guide and individualize such treatment.

Hypocoagulability in this study was observed in only 22% of the dogs, which is much lower than what has been suggested in previous studies, where up to 80% of dogs had variable prolongation of one or more of the routine coagulation tests or thrombocytopenia, believed to be indicative of a hypocoagulant state.29,36,37 The discrepancy between those observations and the results of the TEG analysis can perhaps be explained by the fact that routine coagulation tests are plasma based, whereas the TEG assay is a whole-blood-based assay. Thus, TEG includes both cellular and plasma components important for initiation, amplification, propagation, and lysis of the forming blood clot. The findings indicate that an assay including cellular as well as plasma components, such as TEG, gives a more reliable evaluation of the overall hemostatic state than plasma-based assays alone, with the added benefit that TEG enables overall assessment of hemostasis when the results of plasma assays are ambiguous.

All of the TEG parameters examined in this study are, theoretically, influenced by abnormal hemostasis in both directions and should therefore be affected in both hypo- and hypercoagulable states. Accordingly, the results indicate that even though the dogs, overall hemostatic state was categorized based solely on TEG G value, there were significant differences in k, α, and MA values among hypo-, normo-, and hypercoagulable dogs. Thus as expected, hypocoagulable dogs had prolonged k, lower α and MA compared with normocoagulable dogs, whereas hypercoagulable dogs had shortened k, higher α and MA compared with normocoagulable dogs, indicating that several components of the hemostatic system were affected in these dogs. The results further indicate that it is most likely not necessary to include more TEG parameters when dividing dogs into hypo-, normo-, and hypercoagulable groups.

The 28-day death rate in the TEG hypocoagulable group of dogs was twice that in the hypercoagulable group, indicating that there is a large potential for improvement of therapy in this group of dogs. In both humans and dogs, therapy for DIC is not evidence based and therefore often empirically directed at correcting the imbalance in the hemostatic system, eg, through transfusion of packed red blood cells, fresh frozen plasma (FFP), and heparinization, while treating the underlying disease aggressively. The response to treatment with FFP and heparin is unpredictable and, until now, no laboratory tests have been available to accurately predict or monitor treatment response in these patients. Many drugs modulating hemostasis are available, but limited evidence-based information is available regarding their use in veterinary medicine. TEG may be able to help predict and monitor the response to some of those therapies.

Thirty-three percent of the dogs had normal overall hemostatic capability when evaluated with TF-activated TEG. The death rate in this group of dogs was higher than in the hypercoagulable but lower than in the hypocoagulable dogs, indicating that these dogs might be in a transitory phase between hyper- and hypocoagulable states. Although the dogs included in this study were evaluated with TEG during several days, the average hospitalization was only 3 days, which is unfortunately not long enough to detect significant trends in the TEG profile. Additional longitudinal studies are therefore needed to further characterize this group of dogs with DIC in order to establish whether these dogs are in a transitory phase between hyper- and hypocoagulable states and whether serial measurements will be of benefit to predict outcome.

Another limitation of the study is that the validation of the TF-activated TEG in normal healthy dogs does not account for potential effects of increases in endogenous circulating TF that may influence the results in sick dogs. The relationship between TEG results and endogenous TF activity was not examined in this study, but might be an important area for future research.

Overall, the diversity of the hemostatic changes observed in this group of dogs with DIC emphasizes the complexity of the syndrome and highlights the need for a more differentiated approach to diagnosis and treatment. 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. Thus, future studies should be aimed at providing evidence as to what specific therapy is of benefit to the hypocoagulable and hypercoagulable dogs with DIC and whether the TEG assay will be of value in monitoring patients receiving such therapy.

In conclusion, the TF-activated TEG assay confirmed that overall hemostatic dysfunction is common in dogs with DIC, but most important documented, for the 1st time, that the most frequent abnormality is hypercoagulability and that the case fatality rate was significantly lower in the hypercoagulable than in the hypocoagulable dogs with DIC. The higher mortality rate in the hypocoagulable group is related to many factors, including cause, response to treatment, type of treatment, overall health of the animal, etc., and additional studies are necessary before definitive conclusions can be drawn about mortality rates comparing hyper- to hypocoagulable states. However, the findings suggest that dogs with DIC could benefit from assessment of their overall hemostatic state before supportive therapy guided against DIC and that TF-activated TEG is of value in this regard.

Footnotes

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

aTuman KJ, McCarthy RJ, Patel RB, Ivankovich AD. Quantification of aprotinin reversal of severe fibrinolysis in dogs using thromboelastography. Anesth Analg 1993;76:S439 (abstract)

bMousa S. Synergistic interactions between GPIIb/IIIa antagonists and low molecular weight heparin in inhibiting platelet-fibrin clot dynamics in human blood and in canine model using thromboelastography. Blood 2002;100:3986 (abstract)

cTuman K, Naylor B, Spiess B, et al. Effects of hematocrit on thromboelastography and sonoclot analysis. Anesthesiology 1989;71:A414 (abstract)

dSpiess B, McCarthy R, Ivankovich A. Primary fibrinolysis or D.I.C. differentiated by different viscoelastic tests. Anesthesiology 1989;71:A415 (abstract)

eVacuette, Greiner Bio-One International AG, Kremsmunster, Austria

fTEG 5000 Haemostasis Analyzer, Haemoscope Corporation, Niles, IL

gInnovin, Dade Behring, Marburg, Germany

hACL9000, Instrumentation Laboratory, Warrington, UK

iAdvia 120, Bayer A/S, Lyngby, Denmark

jNycoCard READER II, Medinor A/S, Denmark

kGraphPad Prism v4.01, GraphPad Software, San Diego, CA

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References
  • 1
    Taylor FB Jr., Toh CH, Hoots WK, et al. Towards definition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation. Thromb Haemost 2001;86:13271330.
  • 2
    Toh CH, Downey C. Back to the future: Testing in disseminated intravascular coagulation. Blood Coagul Fibrinolysis 2005;16:535542.
  • 3
    Bick RL, Arun B, Frenkel EP. Disseminated intravascular coagulation. Clinical and pathophysiological mechanisms and manifestations. Haemostasis 1999;29:111134.
  • 4
    Feldman BF, Zinkl JG, Jain NC, Stein SS. Schalm's Veterinary Hematology, 5th ed. Baltimore, MD: Lippincott Williams & Wilkins; 2000.
  • 5
    Hoffman M, Monroe DM III. A cell-based model of hemostasis. Thromb Haemost 2001;85:958965.
  • 6
    McVey JH. Tissue factor pathway. Baillieres Best Pract Res Clin Haematol 1999;12:361372.
  • 7
    Butenas S, Branda RF, Van't Veer C, et al. Platelets and phospholipids in tissue factor-initiated thrombin generation. Thromb Haemost 2001;86:660667.
  • 8
    Rosing J, Van Rijn JL, Bevers EM, et al. The role of activated human platelets in prothrombin and factor X activation. Blood 1985;65:319332.
  • 9
    Hemker HC, Van Rijn JL, Rosing J, et al. Platelet membrane involvement in blood coagulation. Blood Cells 1983;9:303317.
  • 10
    Levi M, De JE, Van Der PT. New treatment strategies for disseminated intravascular coagulation based on current understanding of the pathophysiology. Ann Med 2004;36:4149.
  • 11
    Burstein SA, Peng J, Friese P, et al. Cytokine-induced alteration of platelet and hemostatic function. Stem Cells 1996;14 (Suppl 1):154162.
  • 12
    Osterud B, Flaegstad T. Increased tissue thromboplastin activity in monocytes of patients with meningococcal infection: Related to an unfavourable prognosis. Thromb Haemost 1983;49:57.
  • 13
    Franco RF, De Jonge E, Dekkers PE, et al. The in vivo kinetics of tissue factor messenger RNA expression during human endotoxemia: Relationship with activation of coagulation. Blood 2000;96:554559.
  • 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;34:389393.
  • 15
    Mallett SV, Cox DJ. Thrombelastography. Br J Anaesth 1992;69:307313.
  • 16
    Wiinberg B, Jensen AL, Kjelgaard-Hansen M, et al. Study on biological variation of haemostatic parameters in clinically healthy dogs. Vet J 2007;174:6268.
  • 17
    Otto CM, Rieser TM, Brooks MB, et al. Evidence of hypercoagulability in dogs with parvoviral enteritis. J Am Vet Med Assoc 2000;217:15001504.
  • 18
    Ao H, Moon JK, Tashiro M, et al. Delayed platelet dysfunction in prolonged induced canine hypothermia. Resuscitation 2001;51:8390.
  • 19
    Kang YG, Martin DJ, Marquez J, et al. Intraoperative changes in blood coagulation and thrombelastographic monitoring in liver transplantation. Anesth Analg 1985;64:888896.
  • 20
    Spiess BD, Tuman KJ, Mccarthy RJ, et al. Thromboelastography as an indicator of post-cardiopulmonary bypass coagulopathies. J Clin Monit 1987;3:2530.
  • 21
    Thomson A, Napier JA, Wood JK. Use and abuse of fresh frozen plasma. Br J Anaesth 1992;68:237238.
  • 22
    Lev EI, Ramchandani M, Garg R, et al. Response to aspirin and clopidogrel in patients scheduled to undergo cardiovascular surgery. J Thromb Thrombolysis 2007;24:1521.
  • 23
    Tantry US, Bliden KP, Gurbel PA. Overestimation of platelet aspirin resistance detection by thrombelastograph platelet mapping and validation by conventional aggregometry using arachidonic acid stimulation. J Am Coll Cardiol 2005;46:17051709.
  • 24
    Bakhtiari K, Meijers JC, De Jonge E, et al. Prospective validation of the International Society of Thrombosis and Haemostasis scoring system for disseminated intravascular coagulation. Crit Care Med 2004;32:24162421.
  • 25
    Begg CB. Biases in the assessment of diagnostic tests. Stat Med 1987;6:411423.
  • 26
    Philbrick JT, Horwitz RI, Feinstein AR. Methodologic problems of exercise testing for coronary artery disease: Groups, analysis and bias. Am J Cardiol 1980;46:807812.
  • 27
    Cauchie P, Cauchie C, Boudjeltia KZ, et al. Diagnosis and prognosis of overt disseminated intravascular coagulation in a general hospital – Meaning of the ISTH score system, fibrin monomers, and lipoprotein-C-reactive protein complex formation. Am J Hematol 2006;81:414419.
  • 28
    Wada H, Sakuragawa N, Mori Y, et al. Hemostatic molecular markers before the onset of disseminated intravascular coagulation. Am J Hematol 1999;60:273278.
  • 29
    Bateman SW, Mathews KA, Abrams-Ogg AC, et al. Diagnosis of disseminated intravascular coagulation in dogs admitted to an intensive care unit. J Am Vet Med Assoc 1999;215:798804.
  • 30
    Stokol T, Brooks MB, Erb HN, et al. d-dimer concentrations in healthy dogs and dogs with disseminated intravascular coagulation. Am J Vet Res 2000;61:393398.
  • 31
    Creasey AA, Chang AC, Feigen L, et al. Tissue factor pathway inhibitor reduces mortality from Escherichia coli septic shock. J Clin Invest 1993;91:28502860.
  • 32
    Kienast J, Juers M, Wiedermann CJ, et al. Treatment effects of high-dose antithrombin without concomitant heparin in patients with severe sepsis with or without disseminated intravascular coagulation. J Thromb Haemost 2006;4:9097.
  • 33
    Ten Cate H, Schoenmakers SH, Franco R, et al. Microvascular coagulopathy and disseminated intravascular coagulation. Crit Care Med 2001;29:S95S97.
  • 34
    Griffin A, Callan MB, Shofer FS, et al. Evaluation of a canine d-dimer point-of-care test kit for use in samples obtained from dogs with disseminated intravascular coagulation, thromboembolic disease, and hemorrhage. Am J Vet Res 2003;64:15621569.
  • 35
    Nelson OL, Andreasen C. The utility of plasma d-dimer to identify thromboembolic disease in dogs. J Vet Intern Med 2003;17:830834.
  • 36
    Feldman BF, Madewell BR, O'Neill S. Disseminated intravascular coagulation: Antithrombin, plasminogen, and coagulation abnormalities in 41 dogs. J Am Vet Med Assoc 1981;179:151154.
  • 37
    Maruyama H, Miura T, Sakai M, et al. The incidence of disseminated intravascular coagulation in dogs with malignant tumor. J Vet Med Sci 2004;66:573575.