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[[Image:Coagulation Cascade.jpg|thumb|right|350px|Coagulation cascade. Source: Wikimedia Commons; Author: Joe D (2007)]]
[[Image:Coagulation Cascade.jpg|thumb|right|350px|Coagulation cascade. Source: Wikimedia Commons; Author: Joe D (2007)]]
==Introduction==
==Introduction==
[[Image:LH_Platelet_Histology.jpg|thumb|right|<center><p>'''Platelets'''</p><sup>©RVC 2008</sup></center>]]
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.
References: [[NationWide Laboratories]] [[Image:LH_Platelet_Histology.jpg|thumb|right|<center><p>'''Platelets'''</p><sup>©RVC 2008</sup></center>]]
Normally, [[Normal Mechanisms of Haemostatic Control|haemostastis]] is maintained by three key events:
Normally, [[Normal Mechanisms of Haemostatic Control|haemostastis]] is maintained by three key events:
*'''Primary haemostasis''' involves platelets and the blood vessels themselves and is triggered by injury to a vessel - platelets become activated, adhere to endothelial connective tissue and aggregate with other platelets. A fragile plug is formed which helps to stem haemorrhage from the vessel. Substances released from platelets during primary haemostasis include vasoactive compounds to induce vasoconstriction and other mediators that cause continued platelet activation and aggregation, as well as contraction of the platelet plug. Primary haemostasis ceases once defects in the vessels are sealed and bleeding stops.
*'''Primary haemostasis''' involves platelets and the blood vessels themselves and is triggered by injury to a vessel - platelets become activated, adhere to endothelial connective tissue and aggregate with other platelets. A fragile plug is formed which helps to stem haemorrhage from the vessel. Substances released from platelets during primary haemostasis include vasoactive compounds to induce vasoconstriction and other mediators that cause continued platelet activation and aggregation, as well as contraction of the platelet plug. Primary haemostasis ceases once defects in the vessels are sealed and bleeding stops.
*'''Fibrinolysis''' is the final stage of restoring haemostasis - it prevents uncontrolled, widespread clot formation and breaks down the fibrin within blood clots. The two most important anticoagulants involved in fibrinolysis are antithrombin III (ATIII) and Protein C. The end products of fibinolysis are fibrin degratation products (FDPs).
*'''Fibrinolysis''' is the final stage of restoring haemostasis - it prevents uncontrolled, widespread clot formation and breaks down the fibrin within blood clots. The two most important anticoagulants involved in fibrinolysis are antithrombin III (ATIII) and Protein C. The end products of fibinolysis are fibrin degratation products (FDPs).
Abnormalities can develop in any of the components of haemostasis. Disorders of primary haemostasis include [[Haemorrhagic_Disease_Pathophysiology#Vascular_Fragility|vessel defects]] (i.e. vasculitis), [[Platelet_Abnormalities#Thrombocytopaenia|thrombocytopenia]] (due to decreased production or increased destruction) and [[Platelet Abnormalities|abnormalities in platelet function]] (e.g. congenital defects). These lead to the occurence of multiple minor bleeds and prolonged bleeding times; petechial or ecchymotic [[Haemorrhage|haemorrhages]] may be seen for example, on the skin and mucous membranes, or ocular bleeds may arise. Generally, intact secondary haemostasis prevents major haemorrhage in disorders of primary haemostasis. When secondary haemostasis is abnormal, [[Haemorrhage|larger bleeds]] are frequently seen. Haemothorax, haemoperitoneum, or haemoarthrosis may occur, in addition to subcutaneous and intramuscular haemorrhages. Petechiae and ecchymoses are not usually apparent, as intact primary haemostasis prevents minor capillary bleeding. Examples of secondary haemostatic disorders include [[:Category:Coagulation Defects|clotting factor deficiencies]] (e.g. hepatic failure, vitamin K deficiency, hereditary disorders) and circulation of substances inhibitory to coagulation (FDPs in [[Disseminated Intravascular Coagulation|disseminated intravascular coagulation]], [[Systemic Lupus Erythematosus|lupus]] anticoagulant). If fibrinolysis is defective, thrombus formation and infarctions may result. Thrombus formation may be promoted by vascular damage, circulatory stasis or changes in anticoagulants or procoagulants. For example, ATIII may be decreased. This can occur by loss due to glomerular disease or accelerated consumption in disseminated intravascular coagulation or sepsis.
Abnormalities can develop in any of the components of haemostasis. Disorders of primary haemostasis include [[Haemorrhagic_Disease_Pathophysiology#Vascular_Fragility|vessel defects]] (i.e. vasculitis), [[Platelet_Abnormalities#Thrombocytopaenia|thrombocytopenia]] (due to decreased production or increased destruction) and [[Platelet Abnormalities|abnormalities in platelet function]] (e.g. congenital defects). These lead to the occurence of multiple minor bleeds and prolonged bleeding times; petechial or ecchymotic [[Haemorrhage|haemorrhages]] may be seen for example, on the skin and mucous membranes, or ocular bleeds may arise. Generally, intact secondary haemostasis prevents major haemorrhage in disorders of primary haemostasis. When secondary haemostasis is abnormal, [[Haemorrhage|larger bleeds]] are frequently seen. Haemothorax, haemoperitoneum, or haemoarthrosis may occur, in addition to subcutaneous and intramuscular haemorrhages. Petechiae and ecchymoses are not usually apparent, as intact primary haemostasis prevents minor capillary bleeding. Examples of secondary haemostatic disorders include [[:Category:Coagulation Defects|clotting factor deficiencies]] (e.g. hepatic failure, vitamin K deficiency, hereditary disorders) and circulation of substances inhibitory to coagulation (FDPs in [[Disseminated Intravascular Coagulation|disseminated intravascular coagulation]], [[Systemic Lupus Erythematosus|lupus]] anticoagulant). If fibrinolysis is defective, thrombus formation and infarctions may result. Thrombus formation may be promoted by vascular damage, circulatory stasis or changes in anticoagulants or procoagulants. For example, ATIII may be decreased. This can occur by loss due to glomerular disease or accelerated consumption in disseminated intravascular coagulation or sepsis.
Apart from unusual cases involving vasculitis, there are two causes of defects in primary haemostasis: [[Platelet_Abnormalities#Thrombocytopaenia|thrombocytopenia]] (reduced platelet number), or [[Platelet_Abnormalities#Thrombocytopathia|thrombocytopathia]] (defective platelet function)<sup>2</sup>.
Apart from unusual cases involving vasculitis, there are two causes of defects in primary haemostasis: [[Platelet_Abnormalities#Thrombocytopaenia|thrombocytopenia]] (reduced platelet number), or [[Platelet_Abnormalities#Thrombocytopathia|thrombocytopathia]] (defective platelet function)<sup>2</sup>.
=== 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.
{| class="wikitable"
|+
!
!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.
References: [[NationWide Laboratories]]
===Platelet Number===
===Platelet Number===
The reference range given for platelet number is usually around 200-500x10<sup>9</sup> per litre, although this varies depending on the laboratory equipment used. Clinical signs due to thrombocytopenia are not commonly encountered until the platelet count drops below 50x10<sup>9</sup>/l, when increased bleeding times may be seen. Haemorrhage during surgery becomes a concern with counts lower than 20x10<sup>9</sup>/l, and spontaneous bleeding arises when platelets are fewer than 5x10<sup>9</sup>/l<sup>2</sup>. These cut-offs are lowered if platelet function is concurrently affected by other factors such as the use of non-steroidal anti-inflammatory drugs<sup>1</sup>.
The reference range given for platelet number is usually around 200-500x10<sup>9</sup> per litre, although this varies depending on the laboratory equipment used. Clinical signs due to thrombocytopenia are not commonly encountered until the platelet count drops below 50x10<sup>9</sup>/l, when increased bleeding times may be seen. Haemorrhage during surgery becomes a concern with counts lower than 20x10<sup>9</sup>/l, and spontaneous bleeding arises when platelets are fewer than 5x10<sup>9</sup>/l<sup>2</sup>. These cut-offs are lowered if platelet function is concurrently affected by other factors such as the use of non-steroidal anti-inflammatory drugs<sup>1</sup>.
===Buccal Mucosal Bleeding Time===
===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.
References: [[NationWide Laboratories]]
The buccal mucosal bleeding time is a simple test that gives a rapid assessment of platelet function, providing platelet numbers are normal. If platelet numbers are below 50x10<sup>9</sup>/l, this test should not be performed since the results will be affected by thrombocytopenia, making them unreliable. The small wound inflicted may also not stop bleeding easily.
The buccal mucosal bleeding time is a simple test that gives a rapid assessment of platelet function, providing platelet numbers are normal. If platelet numbers are below 50x10<sup>9</sup>/l, this test should not be performed since the results will be affected by thrombocytopenia, making them unreliable. The small wound inflicted may also not stop bleeding easily.
Acquired platelet function abnormalities can be drug induced, for example by [[NSAIDs|non-steroidal anti-inflammatory drugs]]<sup>2,3</sup>, or can be secondary to [[Uraemia|uraemia]]. Hereditary defects in platelet function also exist, and [[Coagulation_Factor_Deficiency#Von_Willebrand.27s_Disease|von Willebrand's disease]] is the most common of these.
Acquired platelet function abnormalities can be drug induced, for example by [[NSAIDs|non-steroidal anti-inflammatory drugs]]<sup>2,3</sup>, or can be secondary to [[Uraemia|uraemia]]. Hereditary defects in platelet function also exist, and [[Coagulation_Factor_Deficiency#Von_Willebrand.27s_Disease|von Willebrand's disease]] is the most common of these.
The BMBT is influenced by all the aspects of this phase, including vasoconstriction, platelet adherence and plately aggregation. Although this makes BMBT an effective screening test for the vascular/platelet (primary) phase of haemostasis, it also means it is not purely a test for thrombocytopathia, as it is often considered: BMBT depends on an intact vasospastic response and adequate platelet numbers as well as platelet function<sup>3</sup>. BMBT is a fairly crude test, and has been found to be
The BMBT is influenced by all the aspects of this phase, including vasoconstriction, platelet adherence and plately aggregation. Although this makes BMBT an effective screening test for the vascular/platelet (primary) phase of haemostasis, it also means it is not purely a test for thrombocytopathia, as it is often considered: BMBT depends on an intact vasospastic response and adequate platelet numbers as well as platelet function<sup>3</sup>. BMBT is a fairly crude test, and has been found to be normal in some patients with a known platelet function disorder and vice versa<sup>3</sup>. The results of this test therefore should be interpreted with some caution.
normal in some patients with a known platelet function disorder and vice versa<sup>3</sup>. The results of this test therefore should be interpreted with some caution.
=== 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.
==Tests Evaluating Secondary Haemostasis==
==Tests Evaluating Secondary Haemostasis==
Secondary haemostasis describes the formation of a cross linked fibrin meshwork in the blood clot and is dependent on soluble coagulation factors. Abnormalities in secondary coagulation can occur if there are insufficient coagulation factors, inactive coagulation factors or inhibition of factors. To recap, the soluble coagulation factors are traditionally divided into the intrinsic, extrinsic and common pathways.
Secondary haemostasis describes the formation of a cross linked fibrin meshwork in the blood clot and is dependent on soluble coagulation factors. Abnormalities in secondary coagulation can occur if there are insufficient coagulation factors, inactive coagulation factors or inhibition of factors. To recap, the soluble coagulation factors are traditionally divided into the intrinsic, extrinsic and common pathways.
=== 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 tubesPlace tubes in a water bath at 37 (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 temperaturewill have an effect, clotting would be expected within 13 minutes for the dog and 8 minutes for the cat.
References: [[NationWide Laboratories]]
===Activated Clotting Time===
===Activated Clotting Time===
The activated clotting time (ACT) allows rapid evaluation of secondary haemostasis. The ACT is the time taken for 2ml of fresh whole blood to clot in a tube with a contact activator (diatomaceous earth<sup>2</sup>), but an automated analyser can perform a test with a similar principle. The reaction must occur at body temperature to give a reliable indication of haemostatic ability: this can be achieved by the use of a warm water bath, or in an emergency by holding the tubes under an arm. The normal ACT is 90-120 seconds and <75 seconds in dogs and cats respectively<sup>2</sup>.
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 37 C 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.
References: [[NationWide Laboratories]]
The activated clotting time (ACT) allows rapid evaluation of secondary haemostasis. The ACT is the time taken for 2ml of fresh whole blood to clot in a tube with a contact activator (diatomaceous earth<sup>2</sup>), but an automated analyser can perform a test with a similar principle. The reaction must occur at body temperature to give a reliable indication of haemostatic ability: this can be achieved by the use of a warm water bath, or in an emergency by holding the tubes under an arm.
The contact activator used in the ACT test triggers the intrinsic pathway, and so ACT allows assessment of the intrinsic and common pathways. ACT will therefore be prolonged when factors I, II, V, VIII, IX, X, XI or XII are deficient or abnormal, such as in DIC, liver disease, vitamin K antagonist toxicosis or haemophilia A or B<sup>2</sup>. Thrombocytopenia may also increase ACT.
The contact activator used in the ACT test triggers the intrinsic pathway, and so ACT allows assessment of the intrinsic and common pathways. ACT will therefore be prolonged when factors I, II, V, VIII, IX, X, XI or XII are deficient or abnormal, such as in DIC, liver disease, vitamin K antagonist toxicosis or haemophilia A or B<sup>2</sup>. Thrombocytopenia may also increase ACT.
Although the test is simple to perform, interpretation may be challenging. This is because other small fragments involved in the homeostasis of fibrinogen and fibrin are measured by the test in addition to ''bona fide'' fibrin degradation products. In general, an increase in FDP corresponds to increased fibrinolysis. This can be due to a local problem of fibrin generation such as thrombosis, trauma or chronic bleeding, or be related to a systemic process, usually DIC<sup>3</sup>.
Although the test is simple to perform, interpretation may be challenging. This is because other small fragments involved in the homeostasis of fibrinogen and fibrin are measured by the test in addition to ''bona fide'' fibrin degradation products. In general, an increase in FDP corresponds to increased fibrinolysis. This can be due to a local problem of fibrin generation such as thrombosis, trauma or chronic bleeding, or be related to a systemic process, usually DIC<sup>3</sup>.
{{Learning
== 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 and References ==
Laboratory testing: [[NationWide Laboratories]]{{Learning
|literature search = [http://www.cabdirect.org/search.html?rowId=1&options1=AND&q1=%22Coagulation%22&occuring1=title&rowId=2&options2=AND&q2=tes*&occuring2=title&rowId=3&options3=AND&q3=&occuring3=freetext&x=65&y=9&publishedstart=yyyy&publishedend=yyyy&calendarInput=yyyy-mm-dd&la=any&it=any&show=all Coagulation Tests publications]
|literature search = [http://www.cabdirect.org/search.html?rowId=1&options1=AND&q1=%22Coagulation%22&occuring1=title&rowId=2&options2=AND&q2=tes*&occuring2=title&rowId=3&options3=AND&q3=&occuring3=freetext&x=65&y=9&publishedstart=yyyy&publishedend=yyyy&calendarInput=yyyy-mm-dd&la=any&it=any&show=all Coagulation Tests publications]
|flashcards = [[Equine Internal Medicine Q&A 03]]
|flashcards = [[Equine Internal Medicine Q&A 03]]