Kira L. Epstein
Evaluation of Hemostasis
Appropriate clot formation through primary and secondary hemostasis is required to prevent blood loss associated with vascular injury. Equally as important, fibrinolysis and anticoagulants limit the extent of necessary blood clots and prevent clot formation in normal vessels. Imbalance between these systems can lead to excessive bleeding or clotting. Thus evaluation of hemostasis is indicated in horses with clinical signs of bleeding or thrombosis as well as in those with disease conditions that are known to predispose to generalized activation of coagulation and fibrinolysis.
Sample Collection and Processing
Blood samples for coagulation testing should be collected by clean atraumatic venipuncture, and excessive application of vacuum should be avoided to minimize activation of platelets. A vein that has not been used for blood sampling, drug administration, or catheterization and has no evidence of hematoma formation should be used. With repeated or difficult collection, the initial sample can be either discarded or used for other tests. All samples should be inspected, and any tubes with evidence of clot formation should be discarded.
For most coagulation tests, trisodium citrate (1 : 9 ratio of citrate to blood) is the anticoagulant of choice. Multiple citrate concentrations and tube types are available, and some tests require alternate anticoagulants. For most assays, tests should be performed within 4 hours of sample collection, or plasma should be frozen within 1 hour of harvesting.
Test availability varies with the laboratory. Some tests that require rapid testing on whole blood can only be performed in hospitals that operate the testing equipment. Before submitting tests, the veterinarian should check with the testing laboratory to ensure proper sample collection and processing to avoid inaccurate results.
Excessive Bleeding
Excessive bleeding is almost always the result of hypocoagulability. However, hyperfibrinolysis and increased anticoagulant activity, such as may occur during heparin treatment, also have the potential to result in bleeding. Decreased clot formation can be the result of deficient primary hemostasis (formation of a platelet plug) or secondary hemostasis (formation of cross-linked fibrin clot). In some cases, clinical signs can be used to determine which aspect of clot formation is most affected and to choose the subsequent tests. However, the role of platelets and other cells, such as endothelial cells, in secondary hemostasis is now well recognized, with the cell-based model of coagulation representing in vivo coagulation better than the traditional cascade model. Given that primary and secondary hemostasis are intertwined, it may not be possible to clearly differentiate which one is involved in clinical cases of excessive bleeding.
Primary Hemostasis
Clinical Signs
Both low platelet numbers (thrombocytopenia) and impaired function (thrombocytopathia) can affect primary hemostasis. Typically, a defect in primary hemostasis results in bleeding from mucosal surfaces, surgical or traumatic wounds, and sites of venipuncture and catheter placement. Bruising and ecchymoses, as well as hematoma formation at venipuncture and catheter sites, can also occur with abnormalities of primary hemostasis. Petechiae (very small spots of hemorrhage) are a clinical sign associated with thrombocytopenia, but not thrombocytopathia. Bruising, ecchymoses, and petechiae are most visible on the respiratory, gastrointestinal, and urogenital mucosa and on areas with thin, nonpigmented skin. For this reason, the oral, ocular, nasal, and vulvar mucosa, as well as the inner pinnae of the ears, should be thoroughly evaluated during the physical examination.
Buccal Mucosal or Template Bleeding Times
Buccal mucosal and template bleeding times are determined by creating controlled wounds in the oral mucosa or skin, respectively, and recording the time required for bleeding to stop. Thrombocytopenia and thrombocytopathia prolong buccal mucosal and template bleeding times, but unfortunately, the tests have poor reproducibility and high variability that limit their clinical use.
Platelet Count
Platelet counts, which can be performed with an automated hematology analyzer or by manual platelet count with a hemocytometer, are generally included as part of a complete blood count. However, care should be taken in interpreting platelet counts from a complete blood count performed on EDTA-anticoagulated blood because horses are very susceptible to EDTA-induced pseudothrombocytopenia. The latter is caused by in vitro platelet aggregation and results in falsely low platelet counts. Ideally, automated and manual platelet counts should be performed on citrated samples rather than on samples collected into EDTA tubes. However, if a complete blood count is being performed for other reasons and the platelet count is normal, the additional cost of a platelet count on citrated blood can be avoided.
An estimated platelet count can be done quickly with a blood smear evaluated at 100× magnification. At this magnification, the platelet count/µL of blood can be approximated by multiplying the number of platelets seen in one microscope field by 15,000. When an estimate is performed, it is important to check at the feathered edge of the smear for platelet clumping. If clumping is noted, the estimated platelet count may be falsely decreased.
Clinical signs, such as petechiae, bruising, and hematoma formation, are uncommon with platelet counts above 40,000 to 50,000/µL. Spontaneous bleeding is rarely seen until platelet counts fall below 10,000 to 20,000/µL.
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