Platelet Function Testing - PowerPoint PPT Presentation

About This Presentation
Title:

Platelet Function Testing

Description:

Platelet Function Testing John Francis Ph.D. Florida Hospital Center for Hemostasis and Thrombosis, Orlando, FL, USA www.fhitr.com Typical approach to platelet ... – PowerPoint PPT presentation

Number of Views:498
Avg rating:3.0/5.0
Slides: 57
Provided by: JohnFra9
Category:

less

Transcript and Presenter's Notes

Title: Platelet Function Testing


1
Platelet Function Testing
  • John Francis Ph.D.
  • Florida Hospital Center
  • for Hemostasis and Thrombosis,
  • Orlando, FL, USA

www.fhitr.com
2
Typical approach to platelet function testing
  • personal and family bleeding history
  • CBC and platelet count
  • bleeding time or PFA-100

3
Bleeding Time as a screening test of primary
hemostasis
  • screening for congenital and acquired platelet
    dysfunction
  • screening for von Willebrands Disease
  • screening for aspirin (and similar) effects
  • pre-operative risk assessment

4
The Bleeding Time test lacks clinical benefit
CAP and ASCP position statement 1998
  • in the absence of a history, BT does NOT predict
    bleeding associated with surgery
  • a normal BT does NOT exclude possibility of
    surgical bleeding
  • BT CANNOT reliably identify individuals who have
    recently ingested aspirin or who have a
    drug-induced platelet defect.

5
Attempts to simulate the bleeding time in vitro
  • Platelet retention test
  • Machine Bleeding Time
  • Platelet-Stat test
  • Hemostatometer
  • Thrombostat 4000
  • Platelet Function Analyzer (PFA)-100

6
Principle of the PFA-100
  • Collagen/Epinephrine (CEPI) primary screening
    cartridge
  • Collagen/ADP (CADP) differentiates dysfunction
    due to aspirin

7
Comparison of Bleeding Time and PFA-100
20/23 had abnormal platelet aggregation
Francis et al. Platelets 10 132-136,1999
8
Comparison of Bleeding Time and PFA-100
  • overall agreement in 70-80 cases
  • PFA (CEPI) more frequently abnormal
  • PFA more sensitive to aspirin
  • equivalent sensitivity to congenital platelet
    function defects
  • PFA more sensitive to von Willebrand Disease
  • PFA more cost effective

9
Factors that determine closure time in the PFA-100
  • platelet count
  • hematocrit
  • platelet function
  • drug-induced defects
  • other acquired defects
  • congenital defects
  • von Willebrand Factor

10
Effect of aspirin on the PFA-100
11
Relative sensitivity of PFA and BT to von
Willebrand Disease
Fressinaud et al 1998 Cattanep et al 1999 Dean
et al 2000
12
Bleeding time vs PFA-100
Bleeding Time better
PFA-100 better
Von Willebrands Disease
Aspirin ingestion
Congenital platelet receptor disorders
Platelet secretion defects (CEPI)
Platelet secretion defects (CADP)
  • PFA and BT agree in 70-80 cases
  • PFA correlates more closely with aggregometry
  • PFA-100 is more useful in clinical practice

13
Pre-operative hemostatic testing
Pre-op patients (n5649)
Hemostatic workup Platelets, PT, APTT PFA-100
(EPI, ADP)
97 detectable by PFA-100
Koscielny et al Clin Appl Hemost Thromb 10
195-204, 2004
14
Other tests of platelet function
  • Platelet aggregation
  • Optical
  • Impedance
  • VerifyNow
  • Plateletworks
  • Flow cytometry
  • Thromboelastography

15
Platelet aggregation
  • Impedance (lumi) aggregometry

Optical aggregometry
16
Optical aggregometry
Agonist
LIGHT
Light
17
Optical aggregometry
Arachidonic Acid
Collagen
ADP
18
Impedance aggregometry
  • probe inserted in sample
  • electrical current across electrodes
  • platelets form monolayer on probe
  • electrical resistance (impedance) proportional to
    increasing platelet recruitment and aggregation

19
Lumi-aggregometry
Aggregation
Collagen
ATP Release
20
Lumi-aggregometry vs optical aggregometry
  • faster turnaround time
  • less processing of blood sample
  • release easier to assess
  • requires smaller sample (pediatrics)
  • technically easier
  • affected by thrombocytopenia

21
VerifyNow (Ultegra Rapid PFA)
Mixing chamber
Light source
T
Increasing light transmission
GpIIb/IIIa
Fibrinogen
Platelets activated by specific agonist
Fibrinogen-coated beads
Agglutinated beads fall out of suspension
22
Plateletworks
23
Flow Cytometry
24
Applications of flow cytometry
  • platelet activation
  • diagnosis of specific platelet disorders
  • monitoring antiplatelet agents
  • reticulated platelets (thrombopoiesis)
  • platelet-associated antibodies
  • research applications

25
Detection of platelet activation by flow cytometry
P-Selectin (CD62P)
Annexin V
Fibrinogen binding to GpIIb/IIIa
Micro- particles
Activated platelet
RESTING
DETECTION
ACTIVATION
26
Thromboelastography
27
Thromboelastography
Max Amplitude Fibrinogen Platelets
Clotting Rate Fibrinogen Platelets
Clot Time Factor levels Anticoagulants
28
ThromboelastographyEffect of platelets on clot
formation
R 100 90 ? 67.5 45.5 MA 64.0 22.5
29
Anti-platelet effect of aspirin
  • arachidonic acid converted to thromboxane A2 - a
    potent aggregating agent
  • aspirin blocks cyclo-oxygenase-1 (COX-1)

Arachidonic acid
COX
PGG2
ASA
COX
PGH2
TXA2
30
Assessing the anti-platelet effect of aspirin
  • detect surreptitious aspirin intake
  • transfusion medicine
  • pre-op detection of bleeding risk
  • surgery, spinal anesthesia, lithotripsy
  • measure efficacy of aspirin therapy, control
    compliance, identify resistance
  • cardiovascular medicine

31
Aspirin Resistance
  • widespread use of aspirin for prevention of MI
    and stroke (80 million tablets / day)
  • aspirin resistance appears to be common
  • aspirin resistance (or non-compliance) may be
    associated with greater risk of cardiovascular
    death
  • how should aspirin resistance be defined and
    assessed?

32
Frequency of aspirin resistance
33
Possible causes of aspirin resistance
  • inadequate dose
  • non-compliance
  • other routes of platelet activation bypassing the
    COX-1 (aspirin-sensitive) pathway
  • interference with aspirin-binding sites on
    platelets by concomitant NSAID use
  • genetic defect(s) affecting aspirin sensitivity
  • elevated cholesterol
  • method-dependent factors

34
How should Aspirin Resistance be defined?
  • clinical inability of aspirin to protect against
    arterial thrombosis ?
  • failure of aspirin to inhibit platelet function?
  • normal urinary concentration of thromboxane
    metabolites despite aspirin intake ?

35
How should Aspirin Resistance be measured?
  • Platelet function testing
  • PFA-100
  • platelet aggregation in whole blood
  • platelet aggregation in platelet-rich plasma
  • VerifyNow Aspirin Assay
  • thromboelastography
  • Urinary thromboxane A2 metabolites

36
Aspirin resistance in normal volunteers by PFA-100
Francis (unpublished data)
37
Discordance between PFA-100 and platelet
aggregation
  • 325 patients with stable CVD - 325 mg/day
  • platelet aggregation (ADP and AA)
  • resistance (ADPAA) - 6
  • semi-responders (ADP or AA) - 24
  • PFA-100 (CEPI)
  • resistance - 10
  • low concordance between methods for
    aspirin-resistant subjects - 22

38
Aspirin resistance by PFA-100
  • 53 patients on aspirin (100 mg daily) for 20
    prevention of cerebrovascular accidents
  • asymptomatic (no events for 2 yr) 18
  • PFA-100 prolonged in all patients
  • symptomatic (stroke or TIA) 35
  • PFA-100 significantly shorter
  • PFA-100 normal in 12/35

Grundmann et al. J. Neurol 250 63-66, 2003
39
Aspirin resistance by PFA-100Real or due to
elevated vWF?
  • 120 patients on aspirin (75-300 mg daily)
  • 22 (18.3) aspirin-resistant
  • median CADP significantly shorter in
    aspirin-resistant group
  • vWF levels significantly higher in
    aspirin-resistant patients

Harrison et al. ISTH 2003
40
Aspirin resistance by PFA-100Real or due to
elevated vWF?
Chakroun et al. Brit J. Haematol 124 80-85, 2004
41
Aspirin resistance and outcome
Platelet Aggregation
  • 326 patients with stable CAD on aspirin
  • aspirin resistance assessed by platelet
    aggregation gt70 (ADP) and gt20 (AA)
  • 5.2 - aspirin resistant
  • hazard ratio of death, MI or CVA 3.12 (plt0.03)
  • aspirin resistance associated with more than
    three-fold increase in major adverse event rate

Gumm et al JACC 41 961-965, 2003
42
VerifyNow Aspirin Assay
Mixing chamber
Light source
T
Increasing light transmission
GpIIb/IIIa
Fibrinogen
Platelets activated by Arachidonic Acid
Fibrinogen-coated beads
Agglutinated beads fall out of suspension
43
AspirinWorks test for aspirin resistance
  • 11-dehydro-TXB2 is a stable metabolite of TXA2
  • excreted in urine
  • normal levels in patients on aspirin indicate
    resistance
  • measured by ELISA

44
Aspirin resistance and outcome
Eikelboom et al Circulation 105 1650-1655, 2002
Relative Risk of CV death
Range
11-dehydro-TXB2
1.0
lt134
1st quartile
2.0
134 193
2nd quartile
2.5
194 298
3rd quartile
3.5
gt298
4th quartile
pg urinary 11-dehydro-thromboxane B2/mg
creatinine (normal value gt298)
45
Thromboelastography for measuring aspirin
resistance
Heparinized blood Activated FXIII Reptilase Arachi
donic Acid
No aspirin
Aspirin
46
Prevalence of aspirin resistance in coronary
artery disease
  • PFA-100 gt20
  • VerifyNow gt20
  • Urinary dehydro-TXB2 gt20
  • AA-induced aggregation 5
  • TEG Platelet Mapping (AA) lt1

47
Tests predict clinical outcome
  • Laboratory test
  • Bleeding time
  • PFA-100
  • Aggregation
  • VerifyNow
  • Flow cytometry
  • Urinary 11-dehydro-TxB2
  • Predictive of MACE
  • No
  • Yes
  • Yes
  • Yes
  • No
  • Yes

MACE Major Adverse Cardiac Events
48
The ISTH Position
  • Must develop a clinically meaningful definition
    of AR - linking aspirin-dependent lab tests to
    clinical outcome
  • How do we treat AR? No data to show improved
    outcomes from changing therapy
  • Not appropriate to test for AR or to change
    therapy based on current tests

49
Action of clopidogrel (Plavix)
ADP receptors
P2X1
PLC
Ca
cAMP?
Ca ?
  • blocks GpIIb/IIIa activation
  • irreversible effect (7 days)
  • inhibits aggregation to exogenous ADP
  • prevents amplification by other agonists
  • no effect on cyclooxygenase

Aggregation
Release
50
Testing for clopidogrel
  • Platelet aggregation to ADP
  • PFA-100
  • ADP cartridge relatively insensitive
  • New cartridge in development
  • VerifyNow P2Y12 Assay
  • High correlation with PA (5 and 20 µM ADP)
  • Vaosdilator-Stimulated Phosphoprotein (VASP)
    phosphorylation

51
Clopidogrel resistance
Study n Patients Dose Time CR ()
Jeremo 2002 18 PCI 300/75 24 h 28
Gurbel 2003 92 PCI 300/75 24 h 31 - 35
Mueler 2003 105 PCI 600/75 4 h 5 - 11
Kesmarkey 2003 226 CVD 75 - 31
Total 441 5 - 35
Gurbel et al. Curr Pharm Design 12 1261, 2006
52
Possible mechanisms of clopidogrel resistance
  • Platelet count
  • Concomitant medications
  • Genetic polymorphisms
  • Cytochrome P450 (CYP344)
  • P2Y12

53
SummaryPlatelet Function Testing
  • multiple methods available
  • PFA-100 has advantages over bleeding time
  • lumi-aggregometry is more rapid and convenient
    than the optical method
  • near-patient aggregation methods may be
    advantageous in specific situations
  • TEG provides a global assessment of platelet and
    coagulation function

54
SummaryAspirin Resistance
  • aspirin resistance (AR) can be assessed by
    several methods
  • correlation between methods is generally poor
  • AR by PFA-100 partly related to increased vWF
  • overall, AR appears to be associated with worse
    clinical outcomes
  • lack of consensus of how to manage aspirin
    resistance, and whether correction of the
    laboratory defect improves outcome

55
Aspirin and clopidogrel resistanceOngoing
studies
  • ASCET (ASpirin Nonresponsiveness and Clopidogrel
    Endpoint Trial
  • does switching to clopidogrel improve outcomes
    in AR patients?
  • RESISTOR (Research Evaluation to Study
    Individuals who Show Thromboxane Or P2Y12
    Receptor Resistance
  • does modifying antiplatelet therapy prevent
    myonecrosis after PCI in patients with aspirin
    and clopidogrel resistance?

56
Questions?
www.fhitr.com
Write a Comment
User Comments (0)
About PowerShow.com