V T H T 2 3 2 3C L I N I C A L PAT H O L O G Y K R I S T I N C A N G A , R V T

Thrombocytes and Coagulation

A&P: PG. 230LAB PRO: PP. 30-32

(ANTICOAGULANTS) AND 69-73

Reading Assignment

Thrombocyte Formation

Production of platelets = ___________________________

Produced in bone marrow by ___________________________________(TPO) = stimuli for

PPSCs to differentiate into thrombocyte 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

Megakaryocyte

Megakaryocytes

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>WBCsHave 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 RBCsMost 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

T

L

Platelets…

Play a part in both the __________________ and __________________ formation of clots.

Secrete ________________________Form platelet plugsSecrete ____________________Initiate dissolution of blood clots

(“_______________________”)Secrete _______________that attract neutrophils

and monocytes to sites of inflammationSecrete ______________ factors to help maintain

and repair blood vessels

Normal Platelet Values

Canine: 200,000 – 500,000 /µLFeline: 300,000 – 700,000 /µLAll species: ____________ of ____________/µLHorses = ___________normal concentrationsCattle = ____________normal concentrationsAnimals 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 PFsPlatelet _______________ 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

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!!!1. Manufactures most clotting factors2. 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 Hemostasis Testing

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 ______________ hemostasis assess 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 errorManual methods:

1. Platelet estimation during blood film analysis Formula? ALWAYS USE HIGH POWER, _______________________

2. Unopette system & hemocytometer (NOT COMMONLY USED)

Buccal Mucosal Bleeding Time

Tests _____________ hemostasisEvaluates platelet __________ &

_____________(thrombocytopathy, thrombocytopenia)

Evaluates endothelial cell function (__________)

Test can be affected by certain ___________________

BMBT Procedure

1. Place _______________ animal in _____________ recumbency.

2. Use a strip of gauze to tie upper lip back and expose mucosal surface. (Also acts as ______________)

3. Using a Surgicutt® or a Simplate® lancet, create a small wound (~1 mm deep)

4. Remove blood with filter paper at 30-second intervals DO NOT TOUCH SKIN

5. Stop timing when there is no more blood.6. Normal = ________ minutes (canine/feline)

Buccal mucosal bleeding time

Toenail Bleeding TimeAn alternative to BMBTClip toenail just past

quick to cause bleedingKeeping 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 numbersNormal = ___ – ___seconds (canine/feline)

Prothrombin Time (PT)

Evaluates adequacy of factors associated with _______________ and ___________ pathways

Routinely done by ___________Factor XIII activity not evaluatedPlatelet 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 machineFactor XIII activity not evaluatedPlatelet substitute addedNormal: Canine = 9-11 seconds;

Feline = 10-15 seconds

Fibrinogen Assay

Can be done by ___________ or ________________ methods

Only evaluates _________________ concentration

Can use ____________ anticoagulated sampleConcentrations 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 timeClot Retraction TestOne-Stage Prothrombin Time (OSPT)

Used to confirm warfarin toxicity (rodenticide)Activated Partial Thromboplastin Time

(APTT) PIVKA (proteins induced/invoked by vitamin

K absence)d-Dimer and Fibrin Degradation Products

Quick Coagulation Testing

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

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

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

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 causeIf 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 dietDisseminated 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

ThrombocytopathiaMost common cause is inappropriate use of

________________.Can also be caused by:

________________________________ disorders Rare _____________________ problems Certain _______________

Thrombocytopathy: Drugs Causing Platelet Dysfunction

Table 10-3. Drugs Affecting Platelet FunctionAnesthetics General – Halothane

Local - ProcainAntibiotics Cephalosporins – Cefazolin

Penicillins - AmpicillinAnticoagulants HeparinAntihistamines ChlorpheniramineCardiovascular drugs Propanolol, VerapamilFoods and food additives Ethanol, onionsNon-steroidal anti-inflammatory drugs

Aspirin, Phenylbutazone

Oncologic drugs DaunorubicinPlasma Expanders HetaStarch, DextransMiscellaneous drugs Chlorpromazine

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

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 AcidCommonly 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

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

Available as: ______________citrate, or __________________citrate.

Blue topCommonly used in ________________ medicinePrevents 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 AnticoagulantsTable 2-2Name

Mode of Action

Advantages

Disadvantages

Uses

Heparin Antithrombin Reversible, nontoxic

Clumps WBCs, expensive, staining interference

Critical RBC measurements

EDTA Chelates calcium

Best preservative

Irreversible, shrinks cells

Hematology

Oxalates (potassium, Na, lithium)

Chelates calcium

Temporary Variable effects

Coagulation

Citrates (Na, lithium

Chelates calcium

Nontoxic, reversible

Interferes with blood chemistry

Transfusions, coagulation

Fluorides (Na)

Chelates calcium

Inhibits cell metabolism

Interferes w/ enzymatic tests

Preserves blood glucose