Laboratory evaluation of antiplatelet therapy response

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Laboratory evaluation of antiplatelet therapy
response

• Professor Lina Badimon
• CSIC-Institut Català de CiènciesCardiovasculars
• Hospital de la Santa Creu i Sant Pau
• Barcelona, Spain

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Introduction

• Antiplatelet therapy is the cornerstone for the
  treatment of cardiovascular disease
• Several clinical trials have assessed the
  efficacy-bleeding risk associated to antiplatelet
  therapy
• Platelet function monitoring may
• optimise the antiplatelet dosage
• minimise side effects
• reduce thrombotic events

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Cardiovascular effects of aspirin
TXA2
Pro-aggregatory stimuli
Platelet activation and aggregation
TXA2
AA
ASPIRIN
COX-1
Endothelial cell migration and angiogenesis
PGH2
TX-synthase
TXA2
Platelet
TXA2
Contraction and proliferation of SMC

• Inhibition of TX generation is the main effect of
  aspirin treatmenton platelets
• Measurement of TXB2 indicates efficacy of aspirin
  treatment

Modified from Davì G, Santilli F. J Thromb
Haemost. 2006 Oct4(10)2137-9.
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ADP-activation of platelet function
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Methods for platelet function assessment
VerifyNowTM
Chrono-log impedance aggregometry
PlateletworksTM
Born aggregation
Flow cytometry
PFA-100
Multiplate
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Light transmittance aggregometry (Borns method)
PPP
Transmitted light
100
PRP
Transmitted light
0
Platelet aggregates
Transmitted light
70
TRAP thrombin receptor activating peptide PPP
platelet-poor plasma PRP platelet-rich plasma
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Pros for the clinical application of platelet
aggregation

• Historical criterion standard
• In careful hands it is a powerful research tool
  which led to the discovery of platelet
  glycoproteins and platelet signal transduction
  pathways
• It has also been shown to have predictive ability
  on future coronary events

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Cons for the clinical application of platelet
aggregation

• Time consuming and tedious procedure (13 hours)
• Platelet activation during sampling and
  centrifugation
• Platelet assay in a non-physiological milieu
• Dependence on
• platelet count
• fibrinogen concentration
• lipids (interfering with light transmittance)
• Dependence on concentrations of different
  agonists
• Operator expertise

Therefore, it is difficult to arrive at a
consensus definition of an appropriate threshold
for platelet inhibition!
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VerifyNowTM device

• VerifyNow clopidogrel
• VerifyNow GP IIb/IIIa
• VerifyNow aspirin

• Turbidimetric-based optical detection system
• The assay device contains reagents based on
  microbead agglutination technology
• Platelet aggregation inducers
• human fibrinogen-coated beads
• platelet agonist
• No blood handling required

Mixingchamber
Increase in light transmittance with
agglutination of beads rate and extent of change
measured
Lightsource
GP IIb/IIIa integrin
Fibrinogen

Fibrinogen-coated beads
Agglutinated beads fall out of the solution
Platelets in whole blood activated by specific
agonist in mixing chamber
Modified from Michelson AD, et al. Am J Cardiol
2006984N-10N
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VerifyNowTM device

• Pros
• Point of care device
• Whole blood assay
• No handling
• Rapid, simple, small sample volume
• Predictive of outcomes

• Cons
• Comparison with other methods uncertain

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Cone and plate analyser
Cone
0.13mL whole blood

• Platelet disorders
• Aspirin
• Clopidogrel

Well
Apply sheer force(1,800s1 2 minutes)

• Percentage of covered surface
• Average size

Wash and stain
Image analysis
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Cone and plate analyser

• Pros
• Point of care device
• High shear condition
• Whole blood assay
• Rapid and simple
• Higher sensitivity than aggregometry
• Predictive of outcomes

• Cons
• Not well studied
• Comparability with other methods uncertain

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PFA-100 principles
Aperture147µm
Flow
Membrane
Endothelium
Collagen
Collagen
vWF
AgonistADP/epinephrine
Platelet
Shear rate5,0006,000s1
Erythrocyte
Capillary200µm
Vessel wall
PFA-100 cartridge
vWF von Willebrand Factor
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Recorded CTs

• Low CT normal or activated platelets
• High CT platelet inhibition induced by
  platelet disorder or
• medication
• Aspirin prolongs CT-EPI
• GP IIb/IIIa antagonist prolonged CT-EPI and
  CT-ADP
• Clopidogrel insensitive (prolonged EPI?)
• Heparin no influence

CT closure time EPI epinephrine
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PFA-100

• Pros
• Point of care device
• Whole blood assay
• Rapid, simple, small sample volume
• High shear rate condition
• Predictive of outcomes
• No variations in gender and age

• Cons
• Limited to two available cartridges
• Low reproducibility and specificity
• Comparison relative to other methods uncertain
• Dependent on platelet count and haematocrit
• Smoking and blood group variations

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Flow-cytometry receptor expression
Sample of cell suspension ( labelling with
fluorescent antibodies)
Platelet activation markers

• GPIIb/IIIa receptors
• activated state (PAC-1, JonA-PE)
• bound fibrinogen (anti-fibrinogen antibody)
• fibrinogen binding (FITC-fibrinogen)
• CD62 (P-selectin) exposure
• CD63 exposure
• CD40L exposure
• CD41 exposure
• Detection of platelet-leucocyte aggregates
• VASP phosphorylation
• (intracellular signal transduction)

Fluorescence
Scatteredlight
Light source(laser)
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Flow cytometry receptor expression

• Pros
• Measure different platelet populations
  simultaneously
• Whole blood (minimal manipulation)
• Small volumes
• Preparation methods flexible
• fixation or not with PFA
• permeabilisation or not (labelling of
  intraplatelet proteins)
• possible use of different fluorophores
  simultaneously

• Cons
• Specialised laboratories
• Complementary to other methods

PFA parafomaldehyde
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PlateletworksTM

• Compare platelet counts in a control EDTA,
  withplatelet counts in a citrate tube after
  aggregation witheither ADP or collagen
• Platelet aggregation is measured as the loss
  ofsingle platelets

• Pros
• Simple and rapid
• Cheap
• Whole blood
• No handling
• Correlates well with standard aggregometry
• Monitors antiplatelet therapy

• Cons
• GP IIb/IIIa antagonists
• Aspirin
• Clopidogrel

No predictive value for outcomes
EDTA ethylenedia-minetetraacetic acid tube
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Antiplatelet drug efficacy
The definition of laboratory assessment of
antiplatelet drug efficacy largely depends on
the method used to measure platelet function
No standard has been universally accepted
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Conclusions

• Do we need to monitor platelet function?
• controversial issue
• If so, which is the right test of platelet
  function?
• standardisation needed
• fast and reproducible test needed