Investigation of Coagulopathies

Bleeding diatheses can occur as a result of a failure of primary haemostasis (vascular contraction and platelet aggregation) or of secondary haemostasis (the coagulation cascade). The clinical presentation can give some indication as to the nature of the disorder. Petechiation or ecchymoses of the skin or mucosal surfaces, epistaxis, haematuria, melena, intraocular haemorrhage, bleeding after venipuncture or incision, are usually associated with disorders of primary haemostasis for example thrombocytopenia. Haematoma, bleeding into a body cavity, haemarthrosis or delayed bleeding after surgery, are usually associated with an abnormality of secondary haemostasis for example coagulopathy. Consideration of the age of onset, breed, possible previous episodes, recent surgery or trauma, drug therapy, access to toxins and any similar familial history may all be relevant.

In clinic testing for primary haemostasis

Blood Film examination

Examine a fresh air dried blood smear after staining with Diff Quick. Platelet numbers are assessed by counting the mean number of platelets per 100x oil immersion field in the monolayer of the smear (count ten fields over the width of the smear). Assess for the presence of platelet clumps (these may be small or large, light to dark basophilic aggregates usually seen in the feathered edge). When present, platelet clumps may render the manual estimate and the automated count spuriously low. For an estimate of the platelet count, consider that each platelet is equivalent to between 12-20,000/ul. Multiplying the average platelet number per field by 12-20,000 will give an estimate of the platelet count/ul.

	Plarelets per oil inmersion field
Normal	10-25
Mild thrombocytopenia	6-9
Moderate thrombocytopenia	4-5
Marked thrombocytopenia	0-3

When scanning the blood smear, check for the presence of large platelets (shift platelets). These are immature, hyper-reactive platelets that correspond to a regenerative response in the bone marrow to platelet destruction, consumption or loss. Cavalier King Charles spaniels may have large platelets with decreased platelet counts due to inherited macrothrombocytopaenia.

Buccal Mucosal Bleeding Time (BMBT)

Assessment of BMBT is indicated in animals with signs suggesting a disorder of primary haemostasis in which the platelet count is within reference limits. This test evaluates primary haemostasis (platelet adhesion via von Willebrand factor and aggregation to form a platelet plug). It records the time to cessation of bleeding following a standard incision in the buccal mucosa. Use of manual restraint may be possible in dogs but sedation (ketamine and acepromazine) or anaesthesia is required for cats. BMBT is consistently prolonged in thrombocytopenia, severe azotaemia, and von Willebrand’s disease. Drugs which primarily affect platelets, for example aspirin and phenylbutazone, prolong the BMBT.

Normal values: <4.2minutes in the dog; <3.3minutes in the cat.

Clot retraction

The clotted sample should be examined hourly and should reduce to about 50% of its original volume within 2 hours. This is a crude test of platelet function.

In clinic testing for secondary Haemostasis

Whole blood clotting time

This is a crude test of haemostatic function. Prolonged clotting times are associated with abnormalities of the intrinsic pathway and thrombocytopenia (platelet phospholipid is required for clot formation). Normal results do not exclude a coagulopathy. The whole blood clotting time requires the minimum of equipment and can be readily performed in practice but the activated coagulation time is preferred. Method for whole blood clotting time:

• Perform a venipuncture, discard the first 0.25-0.5ml of blood
• Withdraw a further 3-4ml and place 1ml into each of 2 glass tubes
• Place tubes in a water bath at 37º (or as close as possible)
• Start the timer
• Tip the tubes gently to 90 degrees every 30 seconds until the blood has coagulated
• The average coagulation time is calculated. Although the size of tubes and the temperature will have an effect, clotting would be expected within 13 minutes for the dog and 8 minutes for the cat

Activated clotting time

The activated clotting time (ACT) is a more sensitive test than the whole blood clotting time but requires the use of a specially treated tube. The tube is filled, mixed by inversion and placed in a water bath at 37ºC for 45 seconds (cats) and 60 seconds (dogs). It is then tilted at 5 second intervals until the first clot is observed. This is the end point in this test. The ACT is usually <165 seconds in cats and <95 seconds in dogs. It will be prolonged where there is an abnormality of the intrinsic or common pathway and in severe thrombocytopenia.

ACT is not a sensitive test for diagnosing a coagulopathy as a factor must be decreased to <5% of normal to prolong the coagulation time.

Laboratory testing

Coagulation screen

This is the initial recommended test for further investigation of a bleeding disorder. It includes:

Haemogram. RBC count, haemoglobin concentration, haematocrit, MCV, MCH, MCHC, WBC count and differential, platelet count and cellular morphology report

One stage prothrombin time (OSPT). This is prolonged in deficiencies of the extrinsic pathway (such as occurs in rodenticide poisoning; factor VII deficiency/antagonism) and the common pathway. Factors in the common pathway include fibrinogen (I), thrombin (II) and factors V and X

Activated partial thromboplastin time (APTT). This is prolonged in deficiencies of the intrinsic and common pathways. The APPT only becomes prolonged when factor activity is decreased to <30% of normal. The intrinsic pathway includes factors XII, XI, IX, and VIII and is initiated by contact activation. This test is more sensitive than the ACT

Factor XII deficiency is relatively common in cats (DSH, DLH, Siamese, Himalayan) and has been recognized in dogs. Affected animals have markedly prolonged APTT and ACT but do not have a clinical bleeding disorder since factor XII is not essential for normal haemostasis.

Samples required

EDTA, fresh air dried smears. Citrate plasma from the patient and, if possible, from a control.

Sampling

Take blood before initiating therapy. Dispatch the samples immediately to the laboratory. If this is not possible, separate the citrated plasma, decant into a plain tube without anticoagulant (white doughnut) and refrigerate.

Specific additional tests may be recommended, depending upon the results of the initial screen.

Antiplatelet antibodies

Testing for antiplatelet antibodies is not available, immune-mediated thrombocytopenia (IMT) is a diagnosis of exclusion. IMT may be primary, or secondary to infections, myeloproliferative disease, neoplasia and SLE. There is usually a profound thrombocytopenia but normal OSPT and APTT.

Von Willebrand factor

Von Willebrand factor is important for the adhesion of activated platelets and formation of the primary platelet plug in the early stages of haemostasis; it is activated by tissue damage. Canine von Willebrand’s disease is the most common inherited haemostatic disorder, affecting many breeds of dog, including Doberman Pinschers, Scottish terriers, Shetland sheepdogs and Chesapeake Bay retrievers. There is no particular pattern to the expression of the bleeding tendency. For certain breeds there is a genetic test to clarify von Willebrand status. Please contact the laboratory to discuss test availability.

Where genetic testing is not available, assay for von Willebrand factor (citrated plasma) is possible. The administration of DDAVP (desmopressin) raises levels and this can be utilised clinically (for example, to blood donors before harvesting blood).

Haemophilia A (factor VIII)

Haemophilia A (factor VIII deficiency) is reportedly the most common inherited deficiency of secondary haemostasis.

A test for factor VIII deficiency is performed on citrated plasma. Factor VIII is a cofactor in the intrinsic pathway and deficiencies prolong the APTT. It is sex-linked: affected animals are males while females may be carriers. If the deficiency is marked (<1% of normal factor VIII) the animal will suffer from life-threatening bleeding episodes from a young age. There is a moderate form of the disease, recognised particularly in the German Shepherd.

Fibrin degradation products (FDPs)

Assay not available.

D-dimer

D-dimers are a specific product resulting from the lysis of cross-linked fibrin by the fibrinolytic system. An increased D-dimer concentration in plasma indicates that an excess amount of fibrin has been formed within the vascular space and is undergoing fibrinolytic degradation. D-dimer testing has been shown to be sensitive and specific in providing support for a diagnosis of DIC in dogs and is more sensitive than FDP for the same purpose.

Elevations may, however, be seen in any fibrinolytic condition, for example post surgical wound healing and internal haemorrhage.

This test is only validated for dogs. A study to evaluate the concentration of D-dimers in healthy and sick cats, with and without DIC, concluded that the assay had limited value for diagnosis of DIC in this species (Tholen I et al).

Authors & References

NationWide Laboratories