Platelet Function analysis using Multiple Electrode Aggregometry (Multiplate®)

► Multiplate®

platelet function analysis in whole bloodbased on impedance aggregometry

► Multiplate® instrument

5 channels for parallel tests

easy to use Windows XP based software

automatic analysis and documentation

duplicate sensor forinternal quality control

electronic pipetting

► applications

Multiplate is already one of the most widely used platelet

function tests

put the test cell into the measuring position

attach the sensorcable

pipette 300 µl of saline+ 300 µl of blood*

allow 3 minutes for warming

and equilibration

add the activator

after 6 minutes: print the results discard the test cell

* usually hirudin or heparin blood

► Performing the test

► Multiplate® reagents

comprehensive line of dedicated

reagents

TRAP

platelet activation

GpIIb /IIIa receptor

exposure

degranulation

ADP

Arachidonic Acid

Collagen

ArA1

COX

TXA2

TXA2

COLtest

ASPItest

TRAPtest

ADPtestADPtest

PGE1 ADPtest HS (ADP + PGE1)

activation inhibition

GpIIb/IIIa antagonists:

Reopro ® (abciximab)Aggrastat ® (Tirofiban)Integrillin ® (Eptifibatid)

Aspirin ®NSAID

Clopidogrel PrasugrelCangrelor

1 release of arachidonic acid

► Multiplate tests 1/2

rest

ing

plat

elet

activated platelet

► Multiplate tests 2/2

test activation sensitivity not sensitive for

ASPItest arachidonic acid: is converted to TXA2 by platelet-own cyclooxygenase

aspirin, IIb/IIIa antagonists

clopidogrel, vWF

ADPtest ADP: binds onto platelet ADP receptors clopidogrel, IIb/IIIa antagonists

aspirin, vWF

ADPtest HS ADP + prostaglandin E1 (Prostaglandin is a natural inhibitor and enhances the sensitivity of the assay for clopidogrel)

clopidogrel, IIb/IIIa antagonists

aspirin, vWF

TRAPtest TRAP-6 (thrombin receptor activating peptide): TRAP-6 is a potent agonist which mimicks the platelet-activating action of thrombin

IIb/IIIa antagonists vWF, aspirin, clopidogrel (weak effect on TRAPtest)

COLtest collagen: collagen activates platelet and triggers a release of arachidonic acid from the platelet membrane, which is converted to TXA2 by the Cyclooxygenase

aspirin, IIb/IIIa antagonists

clopidogrel, vWF

RISTOtest Ristocetin: vWF dependent platelet activation via the GpIb receptor

Bernard-Soulier syndrome, severe vWD, aspirin

mild vWD

► multiple electrode aggregometry = MEA

test 1+2

- one Multiplate test cell incorporates two independent sensor units.

- the increase of impedance due to the attachment of platelets to the electrodes is detected for each sensor unit separately and transformed to arbitrary aggregation units (AU) that are plotted against time.

- the duplicate sensors serve as an internal control

- during each measurement Pearson´s correlation coefficient of single measurements of the curves assessed by the two electrode pairs and the difference of the two AUCs is calculated. The result is flagged if the values are outside of the acceptance range (correlation coefficient <0.98, difference to the mean curve >20%).

D. Sibbing et al, Thromb Haemost. 2008 Jan;99(1):121-6.

► blood sample for Multiplate analysis

- citrated blood is the typical sample anticoagulant used for platelet function analysis

- citrate complexes approx. 98% of the free calcium in the sample

- however calcium is an important second messenger of platelet activation

- therefore citrate has the potential to disturb platelet function tests

- for Multiplate analysis a tight attachment of platelets onto the impedance sensor is mandatory, which enhances the effect of calcium depletion on this method

- therefore the analysis of blood anticoagulated by hirudin or heparin is recommended

- hirudin blood collection tubes are commercially available from the manufacturer of Multiplate

„Since hirudin and other direct acting thrombin inhibitors do not reduce the concentration of divalent cations in plasma, these can be considered to provide a more physiological environment than the use of sodium citrate. On this basis, we would agree with the recommendation by Dynabyte that the use of hirudin or another direct acting thrombin inhibitor is preferable for measurements of platelet aggregation performed by MEA (multiple eletrode aggregometry = Multiplate).“ A. Johnson, Thromb Haemost. 2008 Jun;99(6):1127-9.

In PRP not all platelets may be present, the most active platelets and larger platelets may have been lost during centrifugation

In PRP platelets are studied in isolation which is NOT their natural situation

In whole blood other blood cells are also present - erythrocytes (a potential source of ADP and ATP and a means of removing adenosine) and leucocytes which also influence platelet aggregation.

S. Heptinstal et al

► Why study platelet function in whole blood rather than in PRP?

TRAPtest ASPItest ADPtest

no platelet inhibition

100 mg aspirin qd

75 mg clopidogrel qd

100 mg aspirin + 75 mg clopidogrel qd

tirofiban (Aggrastat® i.v.)

17 U

134 U139 U

98 U 89 U

31 U

8 U

88 U

17 U

113 U 102 U 89 U

7 U 3 U 3 U

► examples

A.B. *1971

very weak aggregation in all tests due to Glanzman Thrombasthenia

universityclinic

Frankurt / Main Hannover

medical school

Essen university clinic

► near-patient application of Multiplate

► Multiplate in interventional cardiology / neuroradiology

Aggrastat Aggrastat

2 patients before and

after aggrastat loading

dose

case report by Dr. med. Stefanie Müller-Schunk, Neuroradiologie, Klinikum der Universität München

aspirin 500 mg

i.v.

50 min after aspirin

application

case reports by Dr. med. Stefanie Müller-Schunk, Neuroradiologie, Klinikum der Universität München

aspirin 500 mg

i.v.

2 patients before and

after aspirin 500 mg i.v.

clopidogrclopidogrel el 2x75mg2x75mg

clopidogreclopidogrel 75mgl 75mg

Non-response

Response

► increase of clopidogrel dose

Stent thrombosis

0 6 min

AU

Clopidogrel 150mg/d

AU

0 6 min

Prasugrel 10mg/dF. Krötz, Kardiologie, Klinikum der Universität München

► change from clopidogrel to prasugrel

cartridge-basedplatelet

function tests

optical aggregometry

flow cytometry

Platelet Function

Analysis Market

• simple

• whole-blood-based

• 1-2 different tests available

• single chanel

• complicated

• low standardisation

• used in research and reference labs

• easy-to-use

• multi-channel

• several tests available

• open system

Multiplate ®

Multiplate® is one of the newer platelet function tests.

The system has a high sensitivity for anti-platelet drugs (Aspirin, Clopidogrel, Prasugrel, IIbIIIa antagonists).

It is widely used in laboratory and near-patient settings.

Studies have shown a good predictivity towards thromboembolism in platelet non-responsiveness (10 fold enhanced risk for stent thrombosis in a study including 1608 patients following PCI).

Studies have shown a predictivity for transfusion requirements during / after cardiac surgery.

Further applications include the assessment of platelet disorders, drug interactions and animal models.

► Summary