D-Dimers as a Diagnostic Tool for Disseminated Intravascular Coagulation in Dogs

Ashley L. Ayoob, DVM; Bruce E. LeRoy, DVM, PhD; Kenneth S. Latimer, DVM, PhD; Melanie E. Johnson, DVM

Class of 2004 (Ayoob) and Department of Pathology (LeRoy, Latimer, Johnson), College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7388

Introduction

Hemostatic abnormalities have been recognized in veterinary medicine for many years, with documentation dating to 1876.¹ Abnormalities in hemostasis range from coagulopathies with active hemorrhage to sub-clinical prothrombotic states, including disseminated intravascular coagulation (DIC). Recent veterinary medicine studies report a higher incidence of prothrombotic states, with thromboembolic disease and DIC being a common finding in patients admitted to the intensive care unit.² As a result of the varying nature of the disease, clinicians have struggled to accurately diagnose presentations of DIC in the early stages, which may indicate insidious DIC. Because these early stages often go undiagnosed, anti-thrombotic therapy, including heparin, anti-thrombin III replacement, and fresh frozen plasma, may not instituted early enough to prevent mortality. This project will provide an overview of hemostasis, and focus on the use of d-dimers as a diagnostic tool for the early detection of DIC in veterinary medicine.

An Overview of Hemostasis and DIC

Hemostasis requires adequate integrity of the blood vessels, platelet function (aggregation) and number, coagulation proteins, anti-coagulants and their cofactors, as well as platelet inhibitors. Disruption of any of these components results in hemostatic abnormalities, including thrombosis and/or hemorrhage. Primary (congenital) disorders of hemostasis (such as hemophilia A) are well understood, but relatively rare. Secondary (acquired) disruptions of hemostasis, however, are commonly recognized in veterinary medicine.³

One of the well-recognized secondary hemostatic abnormalities is DIC, which contributes to a significant level of morbidity and mortality in critically ill patients. DIC occurs due to systemic, rather than local, activation of coagulation and fibrinolysis, and can be triggered by many causes, some of which are listed in Table 1.^1,4-6 Systemic activation of coagulation leads to microthrombus formation throughout the vasculature. This microthrombosis leads to consumption and degradation of platelets, coagulation proteins, and anti-coagulants as well as causing ischemia, which eventually results in multiple organ failure.

Three stages of DIC, including peracute, acute (fulminant), and chronic, are classically described in the literature, each having varying degrees of clinical signs and hemostatic abnormalities.^1,4,5 Patients experiencing peracute DIC often manifest no overt clinical signs and may have mild to no detectable laboratory abnormalities. Animals in fulminant DIC may have a "classic presentation" of fever, acidosis, hypoxemia, proteinuria, bleeding, shock, and evidence of multiple organ failure with severe and profound laboratory abnormalities.¹ Chronic DIC occurs when there is a constant release of low levels of procoagulants; thus clinical signs may range from none to thrombosis to acute hemorrhage. Anti-thrombotic treatment (heparin) is most effective during the peracute phase; thus, the ability to anticipate DIC in its early stages is crucial to survival.¹ Treatment of fulminant DIC (including transfusion, anti-thrombin III replacement, and heparin) is often futile, with an extremely high morbidity rate despite aggressive therapy.

Diagnosis of DIC is difficult, but can be accomplished via a complete physical exam, knowledge of conditions associated with DIC, and serial laboratory coagulation testing. Many tests are available for evaluation of the coagulation system. Traditionally a series of laboratory tests are performed when DIC is suspected, including a platelet count, examination of a blood smear for schistocytosis, prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), measurement of AT-III concentrations, and detection of fibrin degradation products (FDPs).^3-9 These results should be interpreted with caution, as abnormalities in any of these tests alone is not specific for the diagnosis of DIC. In addition to this administration of hetastarch, dextran, and/or heparin (which are often used as part of the therapeutic regimen) may result in spuriously prolonged clotting times. Recent studies have shown that any of these tests used alone has a low specificity and sensitivity; thus, by themselves they are of little use as a diagnostic aid in DIC.^4,7,8 Although these tests are not specific for diagnosis of DIC alone, they are considered useful and reliable diagnostic tools when evaluated concurrently.⁶ Serial monitoring of laboratory trends in individual patients can be useful when DIC is suspected.

What are FDPs and D-dimers and how do they relate to DIC?

Activation of the coagulation cascade results in increased levels of circulating thrombin and plasmin. Thrombin cleaves fibrinopeptides A and B from fibrinogen, leaving soluble fibrin monomers as the end product (Figure 1). Activation of factor XIII results in polymerization of these fibrin monomers into insoluble cross-linked fibrin clots. Increased levels of circulating plasmin causes clot lysis and degradation of fibrinogen and the soluble fibrin monomers (Figure 2). Plasmin cleaves fibrinogen into fragments X,Y,D, and E, known as fibrinogen degradation products (FDPs). Plasmin also cleaves insoluble cross-linked fibrin polymers into x-oligomers. The main x-oligomers are known as d-dimers.

Figure 1. Reprinted from Veterinary Clinics of North America, Vol. 33, Stokol, Plasma D-dimer for the diagnosis of thromboembolic disorders in dogs, pp. 1419-1435, copyright (2003), with permission from Elsevier.

Figure 2. Reprinted from Veterinary Clinics of North America, Vol. 33, Stokol, Plasma D-dimer for the diagnosis of thromboembolic disorders in dogs, pp. 1419-1435, copyright (2003), with permission from Elsevier.

Laboratory measurement of FDPs has routinely been used in both human and veterinary medicine for many years as a diagnostic aid for DIC. These laboratory tests detect products formed via fibrinogenolysis and/or fibrinolysis, including fibrin monomers and fragments X,Y,D, and E.^6,9,11 Thus, an elevated FDP result indicates the action of plasmin only. Studies in human and veterinary medicine report FDPs to have a sensitivity of 72-74% and a specificity of 84% for the detection of DIC.^6,9 This suggests that FDPs may be an insensitive indicator of DIC when used alone.¹²

D-dimers are produced as a result of plasmin activity on cross-linked fibrin polymers. Thus, d-dimers indicate the activity of both thrombin and plasmin and are specific for fibrinolysis.^9,13 D-dimers are effective in detecting both intravascular and extravascular cross-linked fibrin by-products.^5,7 It has been suggested that D-dimers are a more sensitive and specific diagnostic test for DIC than the traditional FDP assays as they detect the presence of freshly formed fibrin clots and concurrent proteolytic degradation of particulate clots.^5,8,10,11,14

In the last decade laboratory tests detecting the presence of d-dimers have been extensively studied in human medicine. D-dimers are now considered the most reliable test for diagnosis of all stages of DIC in human medicine.^6,7,8 Preliminary research trials have been conducted in veterinary medicine in attempts to validate their use in canine patients. Sensitivities and specificities ranging from 76%-100% and 77%-97% respectively have been reported in both healthy and ill canines.^9,14 However, sensitivity depends on the methodology used.

D-dimer assays are readily available and considered to be economical, non-invasive, rapid, and easy to perform.^4,5,8,11 In addition to this, they can be run on citrated blood, thus decreasing the number of venipunctures and amount of blood drawn from an animal suspected of a coagulopathy.⁹ As stated above, they are a sensitive and specific indicator of DIC. However, it must be kept in mind that d-dimers have been detected in patients with localized thrombosis, hepatic disease, renal disease, rheumatoid arthritis, infection, neoplasia, immune-mediated disease, internal hemorrhage, myocardial infarction, and ischemic stroke, as well as post-operatively.^4,12,13 Thus, they are considered to have an extremely high negative predictive value, but their presence is not pathognomonic for DIC. Additional downfalls to d-dimer testing include that their use is not yet validated in all species and no interassay standard exists, therefore, results cannot be extrapolated between methodologies.⁹

D-dimer Laboratory Analysis

Monoclonal antibodies have been generated which recognize the cross-linked domain of d-dimers as an antigenic target. These antibodies are used in all available d-dimer assays. The majority of assays utitlize human antibodies, however one test has been developed which utilizes canine monoclonal antibodies. Several methodologies are available for the detection of d-dimers and have been widely studied in both human and canine medicine. The following will provide a brief overview of these differing methodologies and their evaluation in veterinary medicine for the diagnosis of DIC.

Agglutination assays, including the use of latex beads and red blood cell (RBC) membrane assays, are widely available. The use of RBC membrane assays have not been validated for use in veterinary medicine, thus the remainder of the discussion will concentrate on latex agglutination (LA) assays.⁹ In a LA assay, latex beads are coated with antibodies which d-dimers then agglutinate with causing a color change. LA assays are qualitative tests, testing for the presence of d-dimers, but give no indication of concentration in the sample. Agglutination tests are advantageous in that they are quick, easy to perform, inexpensive, and require no specialized equipment.^2,9,13 The main disadvantages of agglutination assays include subjectivity to interpretation of results, a lower sensitivity compared with other d-dimer methodologies, and qualitative results only. A study by Stokol which measured d-dimer levels in dogs with DIC as well as normal dogs indicated that a very low, or physiologic, level of d-dimers can be found in normal dogs.¹⁴ Thus, a qualitative assay alone may yield false positive results. Recent studies in veterinary medicine evaluating LA assays report a sensitivity of 100% and a specificity of 97% for the detection of d-dimers.¹⁴

Quantitative tests for d-dimers are available, including enzymatic immunoassays (ELISA) and immunoturbidometric systems. ELISA d-dimer assays have been extensively investigated in both human and veterinary medicine. These assays utilize antibodies linked to either an enzyme or indicator dye. When d-dimers bind with these antibodies a color change occurs. The advantages and disadvantages of this test are similar to LA assays.⁹ While studies show that ELISA d-dimer tests are reliable for use in veterinary medicine, it is indicated that they have a lower sensitivity than LA assays, resulting in an increased number of false positives. Unfortunately the only ELISA d-dimer test that has been studied in veterinary medicine, marketed by AGEN Biomedical, is no longer available.⁹

Immunoturbidometric systems utilize antibody coated beads as well, however the reaction of d-dimers with these beads causes changes in plasma turbidity which is read by an automated analyzer. These tests are generally performed in reference laboratories as they require a high level of technical skill as well as additional laboratory equipment. Thus, these tests results generally have a longer turn around as well as a higher cost to the client.^9,13 The advantages this type of test are a quantitative result as well as automated reading which decreases technical error.⁹ This quantitative result yields the concentration of d-dimers in the sample allowing normal dogs to idealistically be separated from dogs in DIC. Sensitivities ranging from 76-85% and specificities ranging from 65-94.7% in canine patients have been reported. This suggests that immunoturbidometric systems for the detection of DIC are reliable, but appear to be less sensitive and specific than LA assays.

To date, studies indicate that human monoclonal antibodies can serve as a sensitive, but non-specific test for d-dimers in canine patients.^{2,6,9,10,12-14} Several of these studies suggest a preliminary reference interval for quantitative assays in canines.^6,9,14 It is important to keep in mind that there appears to be no interassay standard; thus reference intervals cannot be extrapolated between test modalities.⁹ Table 2 provides a list of specific d-dimer assays whose use has been validated in veterinary medicine.

Conclusion

D-dimer evaluation for the diagnosis of DIC in veterinary medicine shows good promise for the future. However, early research has failed to provide consistent evidence that a definitive diagnosis of DIC can be made based on d-dimer assays alone. Results from recent studies do support the use of d-dimer assays in conjunction with complete physical examination and traditional laboratory assessment of coagulation. In addition to this, d-dimer analysis has an extremely high negative predictor value, which may prove to be instrumental in diagnosis and guidance of anti-thrombotic therapy in the future. D-dimer assay results have been validated to be reliable and accurate, but further research is needed to determine their true sensitivity and specificity in clinical disease.

References

1. Feldman, B. et al.: Recognition and Treatment of Disseminated Intravascular Coagulation. In Bonagura (Ed.), Kirk’s Current Veterinary Therapy XIII. 190-194. Philadephia: W.B. Saunders.

2. Monreal, L.: Editorial: D-dimer as a New Test for the Diagnosis of DIC and Thromboembolic Disease. J. Vet. Intern. Med. 17(6): 757-759; 2003.

3. Fox, P. et al.: Peripheral Vascular Disease. In Ettinger Textbook of Veterinary Internal Medicine. 5th edition (Vol.1). 964-980. Philadephia: W.B. Saunders.

4. Senno, S. et al.: Disseminated Intravascular Coagulopathy (DIC): Pathophysiology, Laboratory Diagnosis, and Management. J. Intensive Care Med. 15(3): 144-158; 2000.

5. Berghaus, G. et al.: Disseminated Intravascular Coagulation: Clinical Spectrum and Established as well as New Diagnostic Approaches. Thrombosis and Haemostasis. 82(2): 706-712; 1999.

6. Caldin, M. et al.: Validation of an immunoturbidometric D-dimer assay in canine citrated plasma. Vet. Clin. Pathol. 29(2): 51-54; 2000.

7. Wada, H. et al.: Hemostatic Molecular Markers Before the Onset of Disseminated Intravascular Coagulation. Am. J. Hemat. 60:273-278; 1999.

8. Bick, R.; Baker, W.: Diagnostic Efficacy of the D-Dimer Assay in Disseminated Intravascular Coagulation (DIC). Thrombosis Research. 65; 785-790; 1992.

9. Stokol, T.: Plasma D-dimer for the diagnosis of thromboembolic disorders in dogs. Vet Clin Small Animal. 33: 1419-1435; 2003.

10. Griffin, A. 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. AJVR. 64(12): 1562-1569; 2003.

11. Dempfle, C.: The Use of Soluble Fibrin in Evaluating the Acute and Chronic Hypercoagulable State. Thrombosis and Haemostasis. 82(2): 673-683; 1991.

12. Nelson, O.; Andreasen, C.: The Utility of Plasma D-dimer to Identify Thromboembolic Disease in Dogs. J. Vet. Intern. Med. 17(6): 830-834; 2003.

13. Rossmeisl, J.: Current Principles and applications of D-dimer analysis in small-animal practice. Vet. Med. March 2003: 224-234.

14. Stokol, T. et al.: D-dimer concentrations in healthy dogs and dogs with disseminated intravascular coagualtion. AJVR. 61(4): 393-398; 2000.

Acknowledgment

"Dubious Dog" by Holly West an acrylic on canvas panel found on her Pooches Gracias website. The copyrighted image is used with permission of the artist.

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