Evaluation of Platelet Function
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Evaluation of Platelet Function Dynamics of Hemostasis Dense Body ADP ATP Serotonin Calcium Microtubular System Plasma Membrane PF Alpha Granules Surface Connecting System Exterior Coat (Glycocalyx) PF Factor V Factor VIII R:ag Fibrinogen Fibronectin β Thromboglobulin 4 Glycogen Dense Tubular System Cytosol Factor XIII Mitochondria 3 (vWF) Adhesion 3 seconds Aggregation 10 seconds Blood Coagulation 5 minutes Tissue Injury Exposure of Collagen Platelet Adhesion Release Reaction Platelet Aggregation Formation of Primary Hemostatic Plug Consolidation of Hemostatic Plug Circulating Platelets ADP Fibrinogen Ca Tissue Factor Small Amounts of Thrombin Platelet Factor 3 (phospholipid) Thrombin Fibrin Fibrinogen Endothelium Defects of Platelet Function Defect Aggregation Response N N N N N N N or N * Primary N N N N + + – – + – N * Anatomy of a Platelet ADP RELEASE + – ++ Plasma Factors Ca ++ Plasma Factors Ca ++ Quantity - 200,000 - 400,000/mm 3 Life Span - 10 days 67% in the circulation 33% pooling Megakaryocyte Spleen Storage and Circulation Disk-shaped cells produced in the megakaryocytes of the bone marrow Bone Marrow What are Platelets? Megakaryocyte Mature Platelet + – + Platelets Factor XIIIa Thromboxane A2 Amplification of Coagulation Cascade or Epinephrine Arachidonic Acid 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 ADP Secondary Primary Secondary Raw data for Ristocetin Cofactor Standard Curve using 1:2, 1:4, and 1:8 dilutions 100 90 80 70 60 50 40 30 20 10 0 1 2 3 4 5 0 Aspirin Effect on Platelets 100 90 80 70 60 50 40 30 20 10 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 Platelet Rich Plasma (PRP) Baseline Light Transmission Increased Light Transmission 100 90 80 70 60 50 40 30 Secondary Response (Release) Primary Response In vivo, platelets participate in primary hemostasis by first adhering, then aggregating at the site of an injured blood vessel. In vitro, platelet aggregation assays use various platelet activators to identify abnormal platelet function and to monitor antiplatelet drug therapy. ADP, collagen, epinephrine, ristocetin and arachidonic acid are reagents commonly used to induce platelet aggregation. Primary Response Is the reversible aggregation of platelets by the aggregating agent. The appearance of a biphasic reaction, showing both primary and secondary response, can occur for some agonists at low concentration. Secondary Response Is the result of enhancement of the initial aggregation process due to release of endogenous ADP and the formation of Thromboxane A 2 . The secondary response is irreversible. Platelet Aggregation 100 90 80 70 60 50 40 30 20 10 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 3 100 90 80 70 60 50 40 30 20 10 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 biphasic Abnormal Response to 10 μM ADP – aggregation < 60% 0 3 Helena offers hemostasis products for all your coagulation needs. For more information or to place an order, call toll free 800-231-5663. ADP Collagen An Educational Service of: 100 90 80 70 60 50 40 30 20 10 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 plasma to 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 586-25 7/10(3) Injection Point Time (minutes) Many drugs can induce platelet function defects, resulting in hemorrhage. The most common mechanisms of inter- ference involve the platelet membrane or membrane receptor sites, and the prostaglandin biosynthetic pathways which are inhibited by aspirin. The arachidonic acid platelet aggregation assay is the only practical way to monitor the effects of aspirin therapy, now widely used to prevent stroke and heart attacks. % Aggregation % Aggregation % Aggregation This wall chart developed in collaboration with Arthur 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 % Aggregation Aspirin Arachidonic acid Cyclo- Oxygenase Dense Body Thromboxane Synthetase TxA 2 Released ADP Platelet Aggregation TxA2 Endoperoxides 100 90 80 70 60 50 40 30 20 10 0 1 2 4 5 6 7 8 9 0 3 Ristocetin – Type I vWD % Aggregation 100 90 80 70 60 50 40 30 20 10 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 % Aggregation 100 90 80 70 60 50 40 30 20 10 0 1 2 4 6 7 8 9 0 3 Ristocetin – Type IIb vWD % Aggregation 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 1.2 0.9 0.6 Platelet Agglutination Helena’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 a standard curve, allowing quantitation of % Ristocetin Cofactor 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. Changes in aggregation are recorded as platelet-rich plasma and aggregating reagents are stirred together in a cuvette. The aggregometer serves as a standardized spectrophotometer. As aggregation proceeds, more light passes through the sample. Platelet Poor Plasma Aggregate Clumping Aggregating Reagent 20 10 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
Transcript of Evaluation of Platelet Function
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
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0
1 2 3 4 50
Aspirin Effect on Platelets
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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
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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
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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
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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
