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  1. Thrombocytes and Coagulation VTHT 2323 Clinical Pathology Kristin Canga, RVT

  2. Reading Assignment A&P: pg. 230 Lab Pro: pp. 30-32 (Anticoagulants) and 69-73

  3. Thrombocyte Formation Production of platelets = ___________________________ Produced in bone marrow by __________ _________________________(TPO) = stimuli for PPSCs to differentiate intothrombocyte precursor. Platelet parent cell =_________________________________

  4. Thrombocyte Formation • Megakaryocyte undergoes incomplete ___________: • (___________________ divides but cytoplasm doesn’t) • Small chunks (~1,000 – 5,000 per megakaryocyte) break off while still in bone marrow, and are sent into circulation as platelets • Some platelets are stored in ______________ for release as needed

  5. Megakaryocytes

  6. Megakaryocyte

  7. Megakaryocytes

  8. Megakaryocytes: Platelet formation Infoldings develop into plasma membrane that divide marginal _______________ into little compartments. These compartments break off and enter bloodstream as ______________. Some platelets are stored in the ___________, while others circulate freely in the blood and live for about ________ days in dogs and just over ___ day in cats.

  9. Megakaryocyte Video http://www.youtube.com/watch?v=6R-ESPFiKbo&feature=related&ajax=1&nocache=1271011451258

  10. Thrombocytes • Commonly referred to as________________. • Not complete cells (lack a ____________), but frequently listed as one of the cell types in peripheral blood. • RBCs>PLTs>WBCs • Have a greater variety of _________________than any of the true blood cells. • Are responsible for _________________phase of clot formation AND initiation of ________________phase of clot formation.

  11. Thrombocyte Morphology • Most are _______________than RBCs • Most PLTs in circulation are _____________ and have numerous, small, purple/pink _____________ scattered throughout the cytoplasm. • Occasionally_________ platelets are seen in blood smear • Giant platelets are considered more _______________ than smaller platelets

  12. Giant Platelet in Peripheral Blood L T

  13. Platelets… Play a part in both the __________________ and __________________ formation of clots. Secrete ________________________ Form platelet plugs Secrete ____________________ Initiate dissolution of blood clots (“_______________________”) Secrete _______________that attract neutrophils and monocytes to sites of inflammation Secrete ______________ factors to help maintain and repair blood vessels

  14. Normal Platelet Values Canine: 200,000 – 500,000 /µL Feline: 300,000 – 700,000 /µL All species: ____________ of ____________/µL Horses = ___________normal concentrations Cattle = ____________normal concentrations Animals will bleed spontaneously if PLT concentration is ≤ ____________ to ____________ /µL

  15. Normal Platelet Morphology

  16. Normal Platelet Morphology

  17. Function of Platelets • Platelets are essential for ________________. • Role of platelets depends on ______________ numbers being present in the blood. • There are 3 main functions of platelets: • 1) Maintenance of __________________ integrity • 2) _________________________ formation • 3) ______________________ of plug by contributing to ______________ formation.

  18. Function of Platelets: 1) Vascular Integrity Platelets attach to _______________________ Release endothelial ____________ factor into endothelial cells. (Prevents leakage of blood in to tissues) ________________ or _______________may occur if there are __________________ numbers of platelets in circulation.

  19. Platelets: 2) Plug Formation • Damaged blood vessel initiates the _________________ process of clot formation. • Platelet _________________ • The platelets adhere ______________ to the damaged vessel AND each other. • __________ often affects this step in the clotting process • Change shape and form ______________ • Allows platelets to intertwine with each other to create platelet __________________. • Platelet ______________________ • The intertwining of platelets to help stop bleeding and causes the release of _________________ factors. • Initiates the _________________ Function of Platelets

  20. Platelets: 2) Plug Formation, cont’d • Release of platelet factors (PFs) that are necessary for the clotting process to be complete. • The aggregation of platelets _______________ the release of PFs • Platelet _______________ occurs after aggregation of platelets. • This is the beginning of the _______________ phase of clot formation.

  21. Platelet Activation • Platelets become activated when there is _____________ to the lining of a blood vessel • The platelets are attracted to the damaged area and stick to it. • Once the platelet has stuck to the damaged vessel, it becomes activated. • Activated platelets have a ______-like appearance and form __________“tails” as they try and catch other platelets. • Dendritic “tails” are sometimes referred to as __________________________

  22. Activated Platelets

  23. Normal Activated Platelets Platelets that have been slightly activated in the sample or by contact with the glass slide (as is common in feline samples) have a stellate form with dendritic processes ("a" in figure). The inset shows a large platelet with centrally aggregated granules which resemble a nucleus.

  24. Platelet Clumping Platelet Clumps Thrombocytosis Platelet Clumps

  25. Platelet Function: 3) Stabilization of Platelet Plug Often referred to as the “______ Matrix” or “Clotting __________” Each step must happen in _________ and ________________ in order for the next step to occur. “____________ Reaction” Converts soluble ______________ to insoluble _________ strands among platelets. Acts as “scaffolding” to encourage ____________.

  26. Fun Video Introduction to Coagulation http://www.youtube.com/watch?v=9QVTHDM90io

  27. Hemostasis • Hemostasis is the process by which blood is prevented from leaking out of _____________ blood vessels. • Depends on three factors: • _____________ of blood vessels • Presence of adequate ___________________ factors • Adequate number of normal circulating ______________. • ___________ is a key player!!! • Manufactures most clotting factors • Bile = essential for _____________ of vitamin ___

  28. Stages of Coagulation • ______________ Hemostasis • _________________________ • Primary _______________ plug formation • Platelet _______________ • Platelet ________________ • Does adhesion or aggregation CAUSE platelet activation?

  29. Stages of Coagulation • _________________ Hemostasis • _________________ Cascade • Ultimate goal = __________ for stabilization of platelet plug • Involves three pathways to clotting: • __________________ Pathway, • __________________ Pathway, and • __________________ Pathway

  30. Stages of Coagulation • Tertiary Hemostasis (________________) • _________ retraction – occurs after ~___ minutes • Platelet Derived ______________ Factor (PDGF) is secreted during clot retraction. • _____________ damage to all tissues involved • Tissue ____________________ Activator is secreted • Clot initiates its own ____________________.

  31. Coagulation Simplified • Extrinsic Clotting Mechanism • chemical outside of blood triggers blood coagulation • triggered by thromboplastin (not found in blood) • triggered when blood contacts damaged tissue • Intrinsic Clotting Mechanism • chemical inside blood triggers blood coagulation • triggered by Hageman factor (found inside blood) • Triggered when blood contacts foreign surface

  32. The Coagulation Cascade

  33. Automated HemostasisTesting • Samples should be collected very carefully with minimal ________________ damage. • ___________ collect sample through indwelling catheters. • Can cause ___________ or blow the vein through manipulation. • Anticoagulant of choice = Sodium citrate • Blocks calcium (but not as strongly as EDTA) • Blue top tube (a.k.a – turquoise) • Results of some testing may be affected by stress, illness, recent exercise, heat cycle (females)

  34. Clotting Tests • Assess one or more of the phases of ________________ (primary, secondary or tertiary) • Tests involving ______________ hemostasisassess intrinsic, extrinsic and/or common pathways. • All patients should undergo coagulation testing prior to undergoing a __________________ procedure. • Platelet estimation • Buccal mucosal bleeding time • Activated clotting time (ACT) • Prothrombin time (PT) • Partial thromboplastin time (PTT) • Fibrinogen assay

  35. Platelet Counting Methods • ____________ or _______________ (least accurate) • Most inaccuracies attributable to • _______________, giant platelets, RBC _____________ • Always use ___________ sample to minimize error • Manual methods: • Platelet estimation during blood film analysis • Formula? • ALWAYS USE HIGH POWER, _______________________ • Unopette system & hemocytometer (NOT COMMONLY USED)

  36. Buccal Mucosal Bleeding Time Tests_____________ hemostasis Evaluates platelet __________ & _____________(thrombocytopathy, thrombocytopenia) Evaluates endothelial cell function (__________) Test can be affected by certain ___________________

  37. BMBT Procedure Place_______________ animal in_____________ recumbency. Use a strip of gauze to tie upper lip back and expose mucosal surface. (Also acts as ______________) Using a Surgicutt® or a Simplate® lancet, create a small wound (~1 mm deep) Remove blood with filter paper at 30-second intervalsDO NOT TOUCH SKIN Stop timing when there is no more blood. Normal = ________ minutes (canine/feline)

  38. Buccal mucosal bleeding time

  39. Toenail Bleeding Time An alternative to BMBT Clip toenail just past quick to cause bleeding Keeping animal undisturbed, monitor for bleeding to cease Normal = <5 minutes (canine/feline)

  40. Activated Clotting Time (ACT) • Evaluates_________________ hemostasis(all factors except Factor VII) • Requires Vacutainer containing sterile _____________________ earth to activate coagulation pathways • 2 mL of blood is collected directly into tube • It is important that tube is pre-warmed and kept at 37º C. • Test can be affected by significantly ______ platelet numbers • Normal = ___ – ___seconds (canine/feline)

  41. Prothrombin Time (PT) Evaluates adequacy of factors associated with _______________ and ___________ pathways Routinely done by ___________ Factor XIII activity not evaluated Platelet substitute added to sample (thrombocytopenia does not interfere) Normal: Canine = 6.4 - 7.4 seconds; Feline = 7 - 11.5 seconds

  42. Partial Thromboplastin Time (PTT) Evaluates adequacy of factors associated with the ___________ and ____________ pathways Routinely done by machine Factor XIII activity not evaluated Platelet substitute added Normal: Canine = 9-11 seconds; Feline = 10-15 seconds

  43. Fibrinogen Assay Can be done by ___________ or ________________ methods Only evaluates _________________ concentration Can use ____________ anticoagulated sample Concentrations may be increased during __________________ or decreased when consumed during coagulation (_________) Normal: Canine = 100 – 250 mg/dL Feline = 100 – 350 mg/dL

  44. Other Coagulation Tests • Whole Blood Clotting time • Clot Retraction Test • One-Stage Prothrombin Time (OSPT) • Used to confirm warfarintoxicity (rodenticide) • Activated Partial Thromboplastin Time (APTT) • PIVKA (proteins induced/invoked by vitamin K absence) • d-Dimer and Fibrin Degradation Products

  45. Quick Coagulation Testing

  46. Coagulopathy • Coagulation defects can be categorized as: • Coagulation defects of primary hemostasis • Coagulation defects of secondary hemostasis • Defects of fibronolysis(tertiary hemostasis)

  47. Coagulation Defects of Primary Hemostasis • Coagulation defects of primary hemostasis • _______________________ or ____________________ (Quantity or quality) • ________________ bleeding • Petechiae, mucosal bleeding, ecchymoses, epistaxis, melena, prolonged bleeding

  48. Coagulation Defects of Primary Hemostasis • ___________________________ • Decreased _______ number • Can be _________________ or _________________ • #1 cause = infectious disease • Ehrlichia, rickettsial diseases, babesiosis, systemic mycoses, toxoplasmosis,mycoplasmosis, Feline retroviruses (FeLV, FIV, FIP), others • Other causes = bone marrow depression; unknown • ______________________________ (vWd) • Decreased or deficient vWF= decreased PLT _____________ , aggregation, and fibrin cross linking • Can occur secondary to______________________ • CS: MM hemorrhage, hematuria, GI bleeding, epistaxis • Screening test of choice = _____________

  49. Defects of Secondary Hemostasis • Coagulation defects of secondary hemostasis • _____________(e.g. pleural, peritoneal, retroperitoneal) • __________________formation • Delayed bleeding/re-bleeding