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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

Thrombocytes and Coagulation

VTHT 2323

Clinical Pathology

Kristin Canga, RVT

reading assignment
Reading Assignment

A&P: pg. 230

Lab Pro:

pp. 30-32 (Anticoagulants)

and 69-73

thrombocyte formation
Thrombocyte Formation

Production of platelets = ___________________________

Produced in bone marrow by __________

_________________________(TPO) = stimuli for PPSCs to differentiate intothrombocyte precursor.

Platelet parent cell =_________________________________

thrombocyte formation1
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
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
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
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

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
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
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
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
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
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
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
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 Clumping

Platelet Clumps


Platelet Clumps

platelet function 3 stabilization of platelet p lug
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
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
Stages of Coagulation
  • ______________ Hemostasis
    • _________________________
    • Primary _______________ plug formation
      • Platelet _______________
      • Platelet ________________
      • Does adhesion or aggregation CAUSE platelet activation?
stages of coagulation1
Stages of Coagulation
  • _________________ Hemostasis
    • _________________ Cascade
      • Ultimate goal = __________ for stabilization of platelet plug
      • Involves three pathways to clotting:
        • __________________ Pathway,
        • __________________ Pathway, and
        • __________________ Pathway
stages of coagulation2
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
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 hemostasis testing
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
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
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 b leeding t ime
Buccal Mucosal Bleeding Time

Tests_____________ hemostasis

Evaluates platelet __________ & _____________(thrombocytopathy, thrombocytopenia)

Evaluates endothelial cell function


Test can be affected by certain ___________________

bmbt procedure
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
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
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
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
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
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
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
  • 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
  • Coagulation defects of primary hemostasis
    • _______________________ or ____________________ (Quantity or quality)
    • ________________ bleeding
    • Petechiae, mucosal bleeding, ecchymoses, epistaxis, melena, prolonged bleeding
coagulation defects of primary hemostasis1
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
Defects of Secondary Hemostasis
  • Coagulation defects of secondary hemostasis
    • _____________(e.g. pleural, peritoneal, retroperitoneal)
    • __________________formation
    • Delayed bleeding/re-bleeding
coagulation defects of secondary hemostasis
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
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
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
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
Qualitative Platelet Dysfunction
  • Thrombocytopathia
  • Most common cause is inappropriate use of ________________.
  • Can also be caused by:
    • ________________________________ disorders
    • Rare _____________________ problems
    • Certain _______________
tertiary hemostatic dysfunctions defective fibrinolysis
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
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
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.

ethylenediamine tetraacetic acid
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
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.