Assessing Platelet Reactivity with Point-of care Systems

Christian Valina Herz-Zentrum Bad Krozingen

Oral Antiplatelet Therapy After PCI: Optimizing Outcomes in Clinical Practice

Assessing Platelet Reactivity with Point-of care Systems

Platelet-function tests

What kind of POC-tests are available?

Differences in test results

Assessing Platelet Reactivity with Point-of care Systems

Platelet-function tests

What kind of POC-tests are available?

Differences in test results

Platelet-function tests

Test principle TestsCessation of bleeding from a standardized wound in vivo

Bleeding time

Aggregometry Light transmission aggregometry (LTA)Multiplate® WBAVerifyNow®

Platelet counting method (e.g. Ichor-plateletworks®)Thrombovision T® guide

Flow cytometry Detection of activation markers ex vivoEx vivo stimulation and detection of activation markersVASP phosphorylation

Shear dependent platelet function within whole blood

PFA-100®

IMPACT® -CPAPlacor PRT-7000®

Global haemostasis tests Thrombelastography (TEG®)Thrombelastometry (ROTEM®)Haemostasis Analysis System®

Thromboxane measurements Serum thromboxane B2

Harrison, Hämostaseologie 2009; 29: 25

Platelet function testing: Some Problems

• No definitive screening test

• Methodological variability within each technique

• Labor intensive

• Costly

• Time consuming

• Requirement of fair degree of expertise and experience to perform and interpret

• Often requirement of additional expensive specialist tests

• Lack of a standardized definition of suboptimal platelet response

Assessing Platelet Reactivity with Point-of care Systems

Platelet-function tests

What kind of POC-Devices are available?

Differences in test results

Point-of-care-testing: What should they can?

• Bed-side testing

• No sample preparation

• Ready-to-use

• Easy to use

• Rapid read out

• Short Turn-around-Time (TAT)

Point-of-care-testing: Test Principles

POC-System Test principle

VerifyNow®

Multiplate® WBAAggregometry

PFA-100®

IMPACT® -CPAPlacor PRT-7000®

Shear dependent platelet function within whole blood

Thrombelastography (TEG®)Thrombelastometry (ROTEM®)Haemostasis Analysis System®

Global haemostasis tests, monitoring of rate and quality of clot formation

Ichor-Plateletworks® Platelet counting pre- and postactivation

HemoStatus® Platelet procoagulant activity

VerifyNow®

• Whole blood (citrated) without further sample preparation

• No need for time-consuming centrifugation steps

• Turbidimetric based optical detection of platelet aggregation stimulated by a specific agonist

• Disposable cartridges contain fibrinogen-coated beads and platelet activator (3 cartridges available: aspirin, P2Y12, GPIIb/IIIa)

VerifyNow® Technology

Results Screen VerifyNow P2Y12 Assay

PRU result is ‘P2Y12-mediated platelet aggregation’ via

adenosine diphosphate (ADP) pathway

VerifyNow® P2Y12 results can then bereported as % platelet inhibition

Base result is ‘Maximal plateletaggregation’ via Thrombin ReceptorActivating Peptide (TRAP) pathwaywhich is independent of aspirin and

clopidogrel

Print

Correlation of VerifyNow® with LTA

0 20 40 60 80 1000

100

200

300

400

500

r2= 0.434p< 0.0001n=562

RPA ADP 5 µM, %

Ve

rify

No

w P

2Y12

PR

U

Multiplate® system (“multiple platelet function analyzer”)

• Whole blood (hirudinized)• Multiple electrodes in the disposable test cell (4 electrodes form 2 independent

sensor units), multiple channels of the instrument (5).• Detects the effects of the platelet inhibitors Aspirin®, clopidogrel and GpIIb/IIIa

antagonists, and also the newer direct ADP receptor antagonists.• No luminescence available, low shear system, does not simulate normal

haemostasis.• Continuously records platelet aggregation.• Increase of impedance by the attachment of Platelets onto the Multiplate® sensors

is transformed to arbitrary aggregation units (AU) and plotted against time. • Three parameters: – Area under the aggregation curve (AUC).– Aggregation is the height of the curve. V– Velocity is the maximum slope of the curve.

Platelet Function Assay 100 (PFA-100)

• Analyzer device and disposable cartridge

• Whole blood

• Measurment of time to occlusion of aperture in mebrane coated with collagen/ADP or collagen/epinephrin under high shear stress conditions

• Cannot distinguish thienopyridene effect

160µm- 40mbar

Flow direction

Thromboelastography (TEG) Haemoscope's TEG® 5000 system

• Small sample of blood (typically 0.36 ml), is placed into a gently rotating cuvette (imitates venous flow and activate coagulation) => Sensor shaft inserted into the sample => a clot forms between the cup and the sensor.

• Speed and strength of clot formation is measured.• Patterns of changes in strength and elasticity in the clot provide information.• Four values that represent clot formation are determined by this test:

– the R value (or reaction time): represents the speed of clot formation – the K value: end or R until the clot reaches 20mm – the angle and

– the MA (maximum amplitude). clot strength • Coagulation Index (CI) (or overall

assessment of coagulability).

Assessing Platelet Reactivity with Point-of care Systems

Platelet-function tests: Advantages of POC-Systems

Platelet-function tests: What kind of POC-tests are available?

Differences in test results

Differences in test results: Are they measuring the same?

• Low shear systems (e.g. Multiplate) does not simulate normal haemostasis

• Different stimulation reagents and dosages

• Is „baseline aggregation“ really „baseline“?

• „Units“:– % platelet inhibition or P2Y12 reaction units (VerifyNow)

– Arbitrary aggregation units (Multiplate)

– Closure time (PFA-100)

– Maximum aggregation, residual aggregation, … (LTA)

– Platelet reacitivity index (VASP-P)

• Cut-off values

Assessing Platelet Reactivity with Point-of care Systems

Platelet-function tests: Advantages of POC-Systems

Platelet-function tests: What kind of POC-tests are available?

Differences in test results

Cut-off values

VerifyNow: Cut off levels for „low response“

• Trenk, 2008; TRIGGER (enrolling) > 208 PRU

• Godino, 2009 > 213 PRU

• Price et al., 2008 ≥ 235 PRU

• ARMYDA-PRO, GRAVITAS (enrolling) ≥ 240 PRU

• Paniccia et al., 2007 ≥ 264 PRU

Assessment of On-Clopidogrel Platelet Reactivity by LTA and VerifyNow

17.6 ± 16.2 %n=307

17.6 ± 16.2 %n=307

161.6 ± 90.5n=307

161.6 ± 90.5n=307

LTALTA VerifyNowVerifyNow

Ver

ifyN

ow

® P

RU

Ver

ifyN

ow

® P

RU

n=307r2=0.441n=307

r2=0.441

Assessment of On-Clopidogrel Platelet Reactivity Correlation Between LTA and VerifyNow

AUC: 0.833Sensitivity for PRU 208: 0.53Specificity for PRU 208: 0.88

AUC: 0.833Sensitivity for PRU 208: 0.53Specificity for PRU 208: 0.88

Assessment of On-Clopidogrel Platelet Reactivity LTA versus VerifyNow by ROC Analysis

Cut off levels for „low response“

• LTA: > 62% (Angiolillo, max. aggregation, ROC)

• TEG: > 50% (5mM ADP)

>70% (10mM, ADP)

• VASP platelet reactivity index: >48%• …..