Thrombocytes and coagulation
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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 __________

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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


  • 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

Giant Platelet in Peripheral Blood




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

Normal Platelet Morphology

Normal Platelet Morphology

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 __________________________

Activated Platelets

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


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


  • 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

The Coagulation Cascade

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


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)

Buccal mucosal bleeding time

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

Quick Coagulation Testing


  • 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

Coagulation Defects of Secondary Hemostasis

  • Congenital clotting factor deficiencies of virtually all known factors have been described. (e.g.: Hemophilia A & B)

  • _____________ coagulation defects can result from:

    • #1 = ________________ toxicity

      • Inhibits vitamin K

      • Vitamin K is required to activate factors II, VII, IX, and X

      • One-step prothrombin time = test to confirm ______________ toxicity.

      • Liver disease, infiltrative bowel disease, and biliary obstruction can also inhibit Vitamin K

Disseminated Intravascular Coagulopathy(DIC)

  • Not a disease in itself; it is a complex _________________ that results from a pathologic condition.

  • Involves __________________ activation of platelets, coagulation proteins, and plasmin; evolving into consumption of coagulation proteins, platelets, and inhibitors of fibrinolysis

  • Some of the many pathologic conditions associated with initiation of DIC include:

    • Trauma and burns

    • Metabolic acidosis/severe shock

    • A large number of infectious diseases

    • _________________________

    • Systemic infection

    • ________________________ disease

    • _________________________

  • Sometimes considered an “_______________ ” condition


  • Laboratory findings are highly _________________

    • Classically ACT, PTT, PT, and thrombin time are prolonged; fibrinogen and platelet counts are decreased

    • _________________________ seen on smear

  • Diagnosis is based on clinical suspicion and at least 3 abnormal coagulation test results.

  • Clinical signs depend on the phase in which the patient is experiencing

    • _____________/Subacute phase: may have few to no overt clinical signs

    • ___________ (consumptive) phase: characterized by venipuncture oozing or modest to severe hemorrhage with inability to form a normal clot

    • ______________ phase: charactized by no clinical signs or oozing of blood

  • Death is caused by extensive microthrombosis or circulatory failure, leading to single or multiple organ failure

Treatment of DIC

  • Successful treatment depends on early detection in critically ill animals.

  • Involves:

    • CORRECTING UNDERLYING _____________________

    • Support of target organs where microthrombi may cause hemorrhage

      • Fluid therapy – balanced electrolyte solutions to maintain effective circulating volume

    • Coagulation factor __________________therapy

    • Administration of _______________ as needed (controversial)

      • Should be accompanied by administration of _____________

    • Close monitoring of antithrombin activity

  • Prognosis is usually _______; depends on underlying cause

  • If an animal survives an acute DIC event, a ___________ form of DIC can exist

Other Acquired Coagulation Defects of Secondary Hemostasis

  • _________________ Disease

    • The __________ synthesizes many of the clotting factors including factors I, II, V, VII, VIII, IX, X, XI, and XII

    • Liver manufacturers __________ which is essential in absorption of vitamin ___ from diet

  • Disseminated Intravascular Coagulation (DIC)

    • A complex syndrome with systemically accelerated coagulation

  • It is clinically difficult to differentiate between hepatic disease and DIC because PT and PTT are usually prolonged with both.

  • DIC can occur secondary to hepatic disease.

Qualitative Platelet Dysfunction

  • Thrombocytopathia

  • Most common cause is inappropriate use of ________________.

  • Can also be caused by:

    • ________________________________ disorders

    • Rare _____________________ problems

    • Certain _______________

Thrombocytopathy: Drugs Causing Platelet Dysfunction

Tertiary Hemostatic Dysfunctions (Defective Fibrinolysis)

The most common dysfunctional state is excessive _____________________. This is an uncommon disease.

Fibrinolysis ______________ can also cause thrombus formation (a condition, not a disease state)

Other Bleeding Disorders

Bleeding disorders may be caused by _____________ or ______________ defects in coagulation proteins, platelets, or vasculature.

Inherited coagulation defects are usually associated with a_________ coagulation protein and often occur at a ____________age.

Acquired coagulation defects often affect________________ coagulation proteins and can occur at _________ age.

Anticoagulants and Hemostasis

As you already know, anticoagulants _____________ or ____________ the formation of a clot.

Anticoagulants are an important part of blood collection.

Different anticoagulants are utilized depending on the _____________ that are needed.

On the following slides, we are going to talk about each anticoagulant and how it can affect your sample.


Heparin is acceptable for most tests requiring ________________. (Green top)

Heparinized tubes should be used for _______________ chemistry analyzers.

Heparin acts on the clotting cascade by preventing the conversion of __________________ to ____________ during the clotting process.

Heparin also may cause _____________of WBCs

Heparin interferes with the _______________ of WBCs and should not be used for blood films.

EthylenediamineTetraacetic Acid

  • Commonly referred to as ___________ (Purple top)

  • Preferred anticoagulant for blood films because it does not alter cell _______________________.

  • Prevents clotting by binding with _______________ , which is necessary for clot formation.

  • Should not be used for ____________ analysis because it affects the metabolism of certain chemicals in the blood and may ____________________ increase or decrease those chemicals.

  • ______________ EDTA can cause shrinkage of _____

    • This will invalidate automated chemistry machines.


Available as: _________oxalate, ______________ oxalate, ______________ oxalate, or ______________oxalate.

_____________________ oxalate is most commonly used. (Grey top)

Prevents clotting by binding with __________, which is necessary for clot formation.

Not frequently used as it interferes with potassium tests, alkaline phosphatase tests, and lactate tests.


Available as: ______________citrate, or __________________citrate.

Blue top

Commonly used in ________________ medicine

Prevents clotting by binding with calcium, which is necessary for clot formation.

Interfere with ___________tests and many of the tests performed by ________________ chemistry analyzers.

Sodium Flouride

Known as __________ preservative but does have anticoagulant properties

Prevents clotting by binding with calcium, which is necessary for clot formation.

May be added to other samples that already have an ______________________.

Also _________top!

Interferes with many _______________ tests performed by automated analyzer.

Commonly Used Anticoagulants

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