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

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

Megakaryocytes


Megakaryocyte

Megakaryocyte


Thrombocytes and coagulation

Megakaryocytes


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

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


Thrombocytes

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

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

Giant Platelet in Peripheral Blood

L

T


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

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


Normal platelet morphology1

Normal Platelet Morphology


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 __________________________


Activated platelets

Activated Platelets


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

Thrombocytosis

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

http://www.youtube.com/watch?v=9QVTHDM90io


Hemostasis

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

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


The coagulation cascade

The Coagulation Cascade


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

      (NOT COMMONLY USED)


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)


Buccal mucosal bleeding time

Buccal mucosal bleeding time


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


Quick coagulation testing

Quick Coagulation Testing


Coagulopathy

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

  • 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


Thrombocytes and coagulation

DIC

  • 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 _______________


Thrombocytopathy drugs causing platelet dysfunction

Thrombocytopathy: Drugs Causing Platelet Dysfunction


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

Heparin

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.


Oxalates

Oxalates

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.


Citrates

Citrates

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.


Commonly used anticoagulants

Commonly Used Anticoagulants


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