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INVESTIGATION OF COAGULOPATHIES IN DOGS
Coagulopathies in dogs most commonly present as a bleeding tendency. This may be due to the failure of primary haemostasis (platelet aggregation and vascular contraction) or to the failure of secondary haemostasis (formation of a platelet/fibrin plug as a result of the coagulation cascade). The history, clinical examination, in-house screening tests and laboratory screening tests all have a place in accurately identifying the abnormality and in initiating therapy to correct the disorder.
The age of onset, breed, history of previous episodes, relation to surgical procedures or trauma, drug therapy, access to toxins and any familial history of haemorrhagic episodes should be recorded.
Coagulopathies due to platelet disorders (primary haemostasis) commonly present with multiple pettechiation or ecchymoses (skin, mucosal surface or gingiva), epistaxis, melaena, haematuria, retinal/intraocular haemorrhage, bleeding after venupuncture or prolonged bleeding from incisions. In contrast coagulation factor defects (secondary haemostasis) commonly present as single haematomata, deep cavity bleeds, haemarthrosis or delayed bleeding/rebleeding. Venupuncture is usually uncomplicated.
IN-HOUSE SCREENING TESTS
1. EXAMINATION OF A STAINED FRESH BLOOD SMEAR
A fresh, air dried blood smear can be rapidly stained (eg Diff Quick stain) and examined under oil immersion. Platelet numbers can be assessed approximately by counting the mean number of platelets per oil immersion field (ten fields over the length of the smear). >5 platelets/oil immersion field corresponds to >100 x 109/L and indicates adequate numbers of platelets. Between 2 and 5 platelets/oil immersion field indicates a thrombocytopenia whilst <2 platelets/oil immersion field indicates a marked thrombocytopenia with a likely bleeding tendency. Additional information which may be noted is the presence or absence of shift (large, immature) platelets. (Examination of RBC and WBC morphology will give an indication of the regenerative status of any anaemia and may indicate the presence of neoplastic cells).
2. THE BUCCAL MUCOSAL BLEEDING TIME TEST
This test utilises a bleeding time device to produce a standardised incision in the buccal mucosa. Sedation or anaesthesia may be required depending on the temperament of the patient. The time to cessation of bleeding is recorded and compared to reference values. It is essentially a screening test for defects in primary haemostasis and is consistently prolonged in cases of thrombocytopenia, severe azotaemia and von Willebrands disease (Jergens et al, 1987). The bleeding time is also prolonged by drug therapy, notably aspirin and phenylbutazone. Both the bleeding time device (Simplate II R) and a protocol for its use are available from the laboratory (LABfaX Test Protocol 5).
3. WHOLE BLOOD CLOTTING TIME AND CLOT RETRACTION
The whole blood clotting time measures the mean length of time taken for freshly drawn blood to clot. A simple protocol involves taking 1ml of blood into each of three glass tubes, inverting every 30 seconds and recording the mean time to clot formation. A normal mean value of 6.1 +/- 0.2 minutes is reported in the literature with the test performed at room temperature (Littlewood, 1992). This test requires adequate platelet numbers (platelet phospholipid) in order to initate clotting and is best interpreted along with a platelet count. A modification of this is the activated clotting time test utilising ciliceous earth vacutainer tubes to initiate clotting. Both the vacutainer tubes and a protocol are available from the laboratory (LABfaX Test Protocol 9).
Clot retraction is assessed by re-examining the tubes after one to two hours. Retraction to 50% of the original volume is considered normal.
Whole blood clotting time tests are prolonged due to failure of the intrinsic clotting pathway and also due to marked thrombocytopenia. The activated clotting time is prolonged when a single factor is depleted to <5% of its normal value. Inadequate clot retraction suggests poor platelet function or thrombocytopenia.
LABORATORY SCREENING TESTS
The Coagulation Screen offered by this laboratory incorporates a haemogram, a one stage prothrombin time (OSPT) test and an activated partial thromboplastin time (APTT) test. Where possible, samples should be taken before initiating therapy using a plastic syringe and tubes. Citrated blood samples should be immediately despatched to the laboratory or should be separated and the plasma refrigerated (< 48 hours) or frozen at -20°C. (The exception is the von Willebrand factor assay which uses citrated whole blood. von Willebrand factor protein is stable for up to 48 hours at 22°C but is adversely affeced by refrigeration).
Evaluation of the haemogram requires an EDTA blood sample and a fresh blood smear. It includes an RBC count, haemoglobin concentration, haematocrit, MCV, MCHC, WBC count and differential, platelet count and a cellular morphology report.
2. OSPT TEST
The OSPT test is performed on citrated plasma and is prolonged due to deficiencies in the extrinsic and common clotting pathways. A time > 3 seconds over the reference range is considered abnormal.
Canine : 7 - 10 seconds
Feline : 7 - 10 seconds
3. APTT TEST
The APTT test is again performed on citrated plasma and is prolonged due to deficiencies in the intrinsic and common clotting pathways. APTT is prolonged due to severe deficiency of a single factor (<30% of its normal concentration).
Canine : 12 - 15 seconds
Feline : 12 - 22 seconds
Sodium citrate tubes are available free of charge from the laboratory. These tubes have a limited shelf life and it is worth checking that they still contain the liquid anticoagulant and that samples are not clotted before dispatch to the laboratory. Tubes should always be filled to the mark (1.3ml) and at least two tubes should be sent for each Coagulation Screen.
Depending upon the results of the Coagulation Screen a number of specific tests may be recommended. These include Anti-Platelet Antibodies (suspected immune-mediated thrombocytopenia), Fibrinogen Degradation Products (suspected disseminated intravascular coagulation), von Willebrand Factor assay and Haemophilia A (Factor VIII) assay. Bone marrow aspiration may be indicated in cases of non-regenerative thrombocytopenia whilst liver function tests may be indicated in suspect hepatopathies.
Abnormalities detected by the OSPTand APTT assays can be further characterised by performing correction tests and specific factor assays. These are beyond the scope of this laboratory and are referred to the Animal Health Trust.
INVESTIGATION OF SELECTED ACQUIRED COAGULOPATHIES
1. IMMUNE MEDIATED THROMBOCYTOPENIA
Destruction of platelets by antibodies is the commonest cause of a marked thrombocytopenia. The condition may be idiopathic or may occur secondary to myeloproliferative disease, lymphoma and systemic lupus erythematosus. It has been associated with drug therapy and can be a sequel to viral infection or vaccination. Typically the bleeding time is increased and the clotting time normal or slightly increased. The time taken for clot retraction is also increased. The OSPT and APTT are both normal. Characteristically there is a profound thrombocytopenia (<50 x 109/L and often <20 x 109/L) (Bush, 1991). Few or no platelets may be present on a fresh smear. Where platelets are present there is often evidence of regeneration (immature or "shift" platetets). Diagnosis may be confirmed by demonstrating the presence of anti-platelet antibodies although often a presumptive diagnosis is made and immunosuppressive therapy initiated.
2. VITAMIN K ANTAGONISM BY COUMARIN DERIVATIVES
Vitamin K antagonism by coumarin rodenticides is one of the more common poisonings seen in dogs. Typically the bleeding time will be normal and the clotting time increased (normal in mild or early cases). Clot retraction will be normal. The OSPT is reliably increased before the APTT (the APTT may be normal in mild or early cases). The platelet count will be normal (Bush, 1991). Samples for the coagulation assays must be taken before vitamin K treatment is initiated. Some of the modern coumarin derivatives have long half-lives and treatment may be required for weeks. If the compound has not been identified it is useful to repeat the OSPT two to three days after cessation of vitamin K therapy to check that the compound is not still active.
Test Code Test Description Sample
COAG Coagulation Screen EDTA, Fresh Smear + Citrate 1:10 (>2ml)
APF Anti-platelet Antibodies EDTA (5ml)
FDP Fibrinogen Degradation Products 1:10 Citrate
VW von Willebrand Factor 1:10 Citrate
BUSH,B.M. (1991). Interpretation of Laboratory Results for Small Animal Clinicians. Blackwell Scientific Publications. Oxford. p 196-219.
JERGENS,A.E.,TURRENTINE,M.A.,KRAUS,K.H AND JOHNSON,G.S. (1987). Buccal mucosa bleeding times of healthy dogs and of dogs in various pathologic states, including thrombocytopenia, uremia, and von Willebrands disease. Am J Vet Res. 48 (9) p 1337-1342.
LITTLEWOOD, J.D. (1992). Differential diagnosis of haemorrhagic disorders in dogs. In Practice 14 (4) p 172-180.
LABfaX Disease Investigation 5 : Version 1 : 01/03/96
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