Thrombocytes and Coagulation VTHT 2323 Clinical Pathology Kristin Canga, RVT
Reading Assignment A&P: pg. 230 Lab Pro: pp. 30-32 (Anticoagulants) and 69-73
Thrombocyte Formation Production of platelets = ___________________________ Produced in bone marrow by __________ _________________________(TPO) = stimuli for PPSCs to differentiate intothrombocyte precursor. Platelet parent cell =_________________________________
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
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.
Megakaryocyte Video http://www.youtube.com/watch?v=6R-ESPFiKbo&feature=related&ajax=1&nocache=1271011451258
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.
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
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
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
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.
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.
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
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.
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 __________________________
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.
Platelet Clumping Platelet Clumps Thrombocytosis Platelet Clumps
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 ____________.
Fun Video Introduction to Coagulation http://www.youtube.com/watch?v=9QVTHDM90io
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 ___
Stages of Coagulation • ______________ Hemostasis • _________________________ • Primary _______________ plug formation • Platelet _______________ • Platelet ________________ • Does adhesion or aggregation CAUSE platelet activation?
Stages of Coagulation • _________________ Hemostasis • _________________ Cascade • Ultimate goal = __________ for stabilization of platelet plug • Involves three pathways to clotting: • __________________ Pathway, • __________________ Pathway, and • __________________ Pathway
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 ____________________.
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
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)
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
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)
Buccal Mucosal Bleeding Time Tests_____________ hemostasis Evaluates platelet __________ & _____________(thrombocytopathy, thrombocytopenia) Evaluates endothelial cell function (__________) Test can be affected by certain ___________________
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)
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)
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)
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
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
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
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
Coagulopathy • Coagulation defects can be categorized as: • Coagulation defects of primary hemostasis • Coagulation defects of secondary hemostasis • Defects of fibronolysis(tertiary hemostasis)
Coagulation Defects of Primary Hemostasis • Coagulation defects of primary hemostasis • _______________________ or ____________________ (Quantity or quality) • ________________ bleeding • Petechiae, mucosal bleeding, ecchymoses, epistaxis, melena, prolonged bleeding
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 = _____________
Defects of Secondary Hemostasis • Coagulation defects of secondary hemostasis • _____________(e.g. pleural, peritoneal, retroperitoneal) • __________________formation • Delayed bleeding/re-bleeding