Tests

 
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Tests for hemostasis generally fall under the categories of primary and secondary hemostasis, fibrinolysis and testing for inhibitors. We have also provided a diagnostic algorithm for test interpretation and a table summaries of thrombocytopenia mechanisms and interpretation of coagulation screening assays. Further information is available on all coagulation tests offered by the Animal Health Diagnostic Center’s Comparative Coagulation Laboratory.

Primary hemostasis

Tests for primary hemostasis involve evaluation of platelet number and function, and assessment of von Willebrand factorIdentification of blood vessel wall problems require the appropriate clinical signs (e.g. skin hyperextensibility with fragility in cats with Ehlers-Danlos syndrome) and histopathologic evaluation of biopsies.

The most common defect in primary hemostasis is acquired thrombocytopenia, therefore it is imperative to obtain a platelet count in every animal with a bleeding disorder.  The only in vivo test for primary hemostasis is the buccal mucosal bleeding time (BMBT), which can also be done in-house, but is considered a more general (global) test of primary hemostasis, since it is affected by defects in vWf, platelet number or platelet function.

Platelets

  • Platelet count and volume: The most common defect in hemostasis is acquired thrombocytopenia, therefore it is imperative to obtain a platelet count in every animal with a bleeding disorder. Platelet numbers can be estimated from smears, in-house, whereas the other tests for primary hemostasis are only offered by referral veterinary laboratories.
  • Platelet function: Platelet function can be assessed through measurement of platelet adhesion, platelet aggregation, and the platelet release reaction. This is obtained through platelet aggregation tests and platelet function analyzers, such as the PFA-100.
  • Global assays (number and function): Buccal mucosal bleeding time (BMBT), clot retraction, whole blood clotting time, platelet function analyzers (PFA). 

    • Whole blood clotting time (WBCT): This is the time taken for blood to clot in glass, plastic or silicone-coated tubes and is dependent on a number of variables. The WBCT in plastic or silicone-coated tubes will be prolonged in thrombocytopenia, however, the results are quite variable and its use is not recommended.
    • Clot retraction: This test measures the amount of time taken for the clot to retact from the sides of a glass container and is dependent upon normal platelet count and function. Clot retraction normally takes between 1-2 hours. This is a very crude test and is not commonly used.

von Willebrand factor

von Willebrand disease (vWD) is the most common inherited defect of hemostasis. Specific assays of canine von-Willebrand factor (vWf) are required for diagnosis of vWD as most dogs with vWD have normal platelet counts and screening coagulation test profiles. The most commonly used method for diagnosis of vWD is measurement of von Willebrand factor antigen (vWf:Ag). Activity of vWf can be assessed in collagen binding assays. The structure of vWf is evaluated through multimeric analysis and defined genetic defects can be detected using DNA-based PCR tests. Global assays such as the BMBT and PFA can also detect vWD, but are less sensitive than more specific assays (and not specific for vWD).

Vessel wall

Identification of blood vessel wall problems require histopathologic evaluation of biopsies. This should be accompanied by the presence of appropriate clinical signs (e.g. skin hyperextensibility with fragility in cats with Ehlers danlos syndrome).

Secondary hemostasis

Tests for secondary hemostasis involve evaluation of coagulation factors and pathways. The routine coagulation screening tests offered by veterinary clinical pathology laboratories are the prothrombin time (PT), activated partial thromboplastin time (APTT), and the thrombin clot time (TCT) or fibrinogen concentration.  The activated coagulation time (ACT) is a quick “in-house” test of the intrinsic and common pathways. The most common cause of abnormalities in secondary hemostasis are acquired conditions, particularly anticoagulant rodenticide toxicosis and DIC. Hemophilia A (factor VIII deficiency) is the most common inherited disorder of secondary hemostasis (vWD is far more common).

Screening assays

  • Activated coagulation time (ACT): A modification of the APTT, the ACT assesses the intrinsic and common pathways in whole blood and requires patient platelets and calcium. It is less sensitive than the APTT but is an available quick in-house screening test.
  • Prothrombin time (PT): The PT measures the activities of extrinsic and common pathway factors
  • Activated partial thromboplastin time (APTT): The APTT is a useful screen for evaluation of the intrinsic and common pathways.
  • Thrombin clot time (TCT): The TCT is a direct measurement of function fibrinogen. Prolonged TCT indicates decreased clot formation and will occur if there is a deficiency or abnormal fibrinogen.
  • Fibrinogen: There are three ways of assessing fibrinogen. These are heat precipitation, clotting fibrinogen and fibrinogen antigen
  • PIVKA: The thrombotest or PIVKA test evaluates proteins induced by vitamin K absence/antagonism (PIVKA).  These proteins are not activated in the presence of vitamin K antagonists or vitamin K deficiency, resulting in a prolonged thrombotest, in which clot formation is dependent on vitamin K dependent factors.

Specific factor analysis

There are currently two methods to determine specific factor activities. The first is based on the ability of the patient plasma to correct the prolonged clotting times of plasma that is deficient in specific factors; the second on the ability of specific factors in patient plasma to cleave chromogenic substrates.

Global assays

These are assays that evaluate thrombin generation or fibrin formation in the presence of cells (platelets or other cellular sources of phospholipids) and includes viscoelastic clot detection methods (thromboelastometry, thromboelastrography) and thrombin generation assays.

Fibrinolysis

Tests for fibrinolysis involve evaluation of the fibrinolytic pathway. Unfortunately, measurement of many components of the fibrinolytic pathway, including plasminogen, tissue plasminogen activator, plasminogen activator inhibitor, are not offered routinely, which is a major shortcoming when evaluating animals for hemostatic or thrombotic disorders. The most common tests of fibrinolytic activity are measurement of lytic products, including FDPs and D-dimer.

  • Products of degradation: FDPs, D-dimer
  • Protein activity or concentration: Measurement of tissue plasminogen activator, plasminogen concentrations. This is really only done in research laboratories and not offered routinely as a diagnostic test.
  • Global assays: Clot lysis time, euglobin lysis test, tPA-induced fibrinolysis using viscoelastic methods.
    • Clot lysis time: This is the time required for whole blood clots to lyse at 37ºC (and can be performed after the clot retraction time) and is dependent on plasmin activity, fibrinogen concentration and the degree of clot retraction. Poor clot retraction will impede the lysis time. The clot is usually lysed within 8 to 20 hours in animals.
    • Euglobulin lysis time: This is a measure of plasmin activity. In this test, euglobulins (fibrinogen, plasminogen, plasmin, plasminogen activator) precipitate on dilution with water, whereas fibrinolytic inhibitors (plasminogen activator inhibitor, antiplasmin) do not. The removal of these inhibitors allows the plasmin in the sample to lyse the precipitate with the time taken for lysis being the euglobulin lysis time. This test is quite crude and is not offered by most laboratories.

Inhibitors

The most commonly used test for inhibitors is measurement of antithrombin (AT) and protein C activities. Tests for other inhibitors, such as α2-antiplasmin, protein S, and heparin cofactor II, are either not readily available, have not been validated for use in animals or are used for research purposes by specific institutions.

  • Secondary hemostasis: AT, protein C activities are measured on their ability to inhibit factor X (AT) or cleave a specific substrate (protein C).
  • Fibrinolysis: Measurement of plasminogen activator inhibitor is not readily available. A method for detection of thrombin activatable fibrinolytic inhibitor was recently tested in dogs.
  • Therapeutic heparin monitoringThis can be done by prolongation of the APTT or measurement of anti-FXa activity, depending on the type of heparin.
  • Anticoagulant rodenticide screens: High performance liquid chromatography.
  • Acquired inhibitors (e.g. lupus anticoagulants, anti-factor VIII antibodies): The presence of acquired inhibitors of secondary hemostasis can be suspected when coagulation tests (usually the APTT) shorten when the patient sample is diluted with sterile saline. In patients with factor deficiencies, the APTT will lengthen due to further dilution of the factors, whereas the APTT will shorten in patients with inhibitors due to dilution of the inhibitors. Specific assays for such inhibitors include Bethesda assays for anti-factor antibodies and modified phospholipid assays, such as the dilute Russell’s viper venom time for measurement of lupus anticoagulant.
  • Factor-inhibitor complexes: High concentrations of thrombin-antithrombin complexes are a sensitive and specific indicator of thrombin activation. The assay appears to detect thrombin-antithrombin complexes in some species and has been used to identify hypercoagulability in specific research projects in dogs, cats and horses. Similarly, high concentrations of plasmin-antiplasmin complexes are a good indicator of plasmin activation, however once again this is not a readily available test. Both thrombin-antithrombin and plasmin-antiplasmin complexes are high in human patients with DIC and these tests could be useful in animals, however none are currently commercially available for veterinary use (likely due to the high cost due to the ELISA methods employed to detect these complexes).

Global hemostasis tests

These can measure multiple pathways simultaneously, including the contribution of cells (platelets, erythrocytes). They include:

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