Evaluation of Platelet Function

Dynamics of Hemostasis

Dense BodyADPATP

SerotoninCalcium

Microtubular System

Plasma MembranePF

Alpha Granules

SurfaceConnecting System

Exterior Coat(Glycocalyx)

PFFactor VFactor VIII R:ag

FibrinogenFibronectinβ Thromboglobulin

4

Glycogen

Dense Tubular System

CytosolFactor XIII

Mitochondria

3

(vWF)

Adhesion3 seconds

Aggregation10 seconds

Blood Coagulation5 minutes

Tissue InjuryExposure of Collagen

Platelet Adhesion

Release Reaction

Platelet Aggregation

Formation ofPrimary Hemostatic Plug

Consolidation ofHemostatic Plug

Circulating Platelets

ADP Fibrinogen Ca

Tissue Factor

Small Amountsof Thrombin

Platelet Factor 3 (phospholipid)

Thrombin

Fibrin

Fibrinogen

Endothelium

Defects of Platelet FunctionDefect Aggregation Response

N

N

N

N

N

N

N or

N *

Primary

N

N

N

N

+

+

–

–

+–N

*

Anatomy of a Platelet

ADPRELEASE

+–

++

Plasma FactorsCa++

Plasma FactorsCa++

Quantity - 200,000 - 400,000/mm3

Life Span - 10 days

67%in the

circulation

33%pooling

Megakaryocyte Spleen

Storage and CirculationDisk-shaped cellsproduced in themegakaryocytes ofthe bone marrow

BoneMarrow

What are Platelets?

Megakaryocyte

Mature Platelet

+–

+

PlateletsFactor XIIIa

Thromboxane A2

Amplification ofCoagulation Cascade

or

Epinephrine ArachidonicAcid Collagen Thrombin Ristocetin

= Normal= Not diagnostic= Corrected by cryoprecipitate, factor VIII concentrate or normal plasma; type IIB exhibits increased sensitivity to low concentrations= Decreased

( Type IIb)

+–

N

N

N or

ADPSecondary Primary Secondary

Raw data for Ristocetin Cofactor Standard Curve using 1:2, 1:4, and 1:8 dilutions

100908070605040302010

0

1 2 3 4 50

Aspirin Effect on Platelets

100908070605040302010

0

1 2 4 5 6 7 8 9

Normal Response – aggregation > 60% with < 1.2 mg/mL ristocetin; response decreases proportionately with 0.9 and 0.6 mg/mL ristocetin

0 3

Ristocetin

PlateletRichPlasma(PRP)

Baseline LightTransmission

Increased LightTransmission

10090807060504030

Secondary Response(Release)

Primary Response

In vivo, platelets participate in primary hemostasis by firstadhering, then aggregating at the site of an injured bloodvessel. In vitro, platelet aggregation assays use variousplatelet activators to identify abnormal platelet function and tomonitor antiplatelet drug therapy. ADP, collagen, epinephrine,ristocetin and arachidonic acid are reagents commonly used toinduce platelet aggregation.

Primary ResponseIs the reversible aggregation ofplatelets by the aggregating agent.The appearance of a biphasicreaction, showing both primaryand secondary response, can occur for some agonistsat low concentration.

Secondary ResponseIs the result of enhancement ofthe initial aggregation process dueto release of endogenous ADPand the formation of Thromboxane A2. The secondary response isirreversible.

Platelet Aggregation

100908070605040302010

0

1 2 4 5 6 7 8 9

Normal Response to 10 µg Collagen – lag phase < 60 seconds Abnormal Response to 10 µg Collagen – prolonged lag phase

0 3

100908070605040302010

0

1 2 4 5 6 7 8 9

Normal Response to 10 µM ADP – Primary and Secondary slopes indistinguishable, aggregation > 60%Normal Response to 5 µM ADP – Primary and Secondary slopes distinctly biphasicAbnormal Response to 10 µM ADP – aggregation < 60%

0 3

Helena offers hemostasis products forall your coagulation needs.

For more information or to place an order,call toll free 800-231-5663.

ADP

Collagen

An Educational Service of:

100908070605040302010

0

1 2 4 5 6 7 8 9

Normal Response to 500 µg Arachidonic Acid – aggregation > 60% Abnormal Response Due to Aspirin

0 3

Arachidonic Acid

The Ristocetin Cofactor Assay measures the ability of a patient’s plasmato agglutinate formalin-fixed platelets in the presence of ristocetin. The rate of ristocetin-induced agglutination is related to the concentration of von Willebrand factor and the percent normal activity can be obtained from the standard curve.

0

Platelet Aggregation

586-257/10(3)

Injection Point

Time (minutes)

Many drugs can induce platelet function defects, resultingin hemorrhage. The most common mechanisms of inter-ference involve the platelet membrane or membranereceptor sites, and the prostaglandin biosynthetic pathwayswhich are inhibited by aspirin. The arachidonic acid plateletaggregation assay is the only practical way to monitor theeffects of aspirin therapy, now widely used to prevent strokeand heart attacks.

% A

ggre

gatio

n %

Agg

rega

tion

% A

ggre

gatio

n

This wall chart developed in collaboration withArthur P. Bode, Ph.D.East Carolina School of Medicine, Greenville, NC

Albert Pattison, B.Sc.Helena Laboratories, (UK) Ltd., Newcastle, England

Helen Ridgway, Ph.D.Wadley Institutes, Dallas, TX

% A

ggre

gatio

n

AspirinArachidonic acid

Cyclo-Oxygenase

DenseBody

ThromboxaneSynthetase

TxA2Released

ADP

PlateletAggregation

TxA2

Endoperoxides

100908070605040302010

0

1 2 4 5 6 7 8 90 3

Ristocetin – Type I vWD

% A

ggre

gatio

n

100908070605040302010

0

1 2 4 5 6 7 8 9

Normal Response to 10 µM Epinephrine – Primary and Secondary slopes indistinguishable, aggregation > 60%Normal Response to 2 µM Epinephrine – Primary and Secondary slopes distincly biphasic; aggregation > 60%Abnormal Response to 10 µM Epinephrine – aggregation < 60%

0 3

Epinephrine

% A

ggre

gatio

n

100908070605040302010

0

1 2 4 6 7 8 90 3

Ristocetin – Type IIb vWD

% A

ggre

gatio

n

Response of Type I von Willebrand’s – aggregation < 60% with 1.2 mg/mL ristocetin; response decreases proportionately with 0.9 and 0.6 mg/mL ristocetin

Response of Type IIb von Willebrand’s – aggregation > 60% with 1.2 mg/mL ristocetin; increased response to 0.9 and 0.6 mg/mL ristocetin

5

1.2

0.9

0.6

1.2

0.9

0.6

1.20.90.6

Platelet AgglutinationHelena’s AggRAM couples 4-channel laser optic modules with a powerful, easy-to-use Microsoft® windows interface to make platelet aggregation and ristocetin cofactor testing easier and more efficient.

Screen at the point-of-care in just 2 minutes using a standard cell counter

• Flexible, modular design • On-screen prompts for procedures

• Quick access to data input screens • Display & print lag phase

• Auto slope & max % aggregation • Tri-level password protection

• Barcode & bi-directional LIS capable • Integral QC action log

• Platelet Aggregation

• Platelet Inhibition

• Platelet Count

AggRAM

Patient values are determined by comparison to astandard curve, allowing quantitation of % RistocetinCofactor Activity.

Standard Curve

Ristocetin Cofactor – Raw Data

1:2

1:4

1:8

The platelet aggregation procedure is performed on a turbidi-metric aggregometer as first described by Born. Changesin aggregation are recorded as platelet-rich plasma andaggregating reagents are stirred together in a cuvette. Theaggregometer serves as a standardized spectrophotometer. Asaggregation proceeds, more light passes through the sample.

PlateletPoorPlasma

AggregateClumping

AggregatingReagent

2010 Lag

+

Typical Biphasic Pattern

ADP • Epinephrine • Collagen • RIPA • Ristocetin Cofactor ADP • Collagen • Arachidonic Acid

1. Bernard-Soulier Syndrome

2. von Willebrand’s Disease

3. Glanzmann’s Thrombasthenia

4. Storage Pool Disorder

5. Aspirin-like Disorder or Aspirin Ingestion

586-25Rev3 plates.pdf 1 7/20/10 8:51 AM