Platelet Function analysis using Multiple Electrode Aggregometry (Multiplate )

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Platelet Function analysis using

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► _Multiplate

®

platelet function analysis

in whole blood

based on

impedance

aggregometry

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► _Multiplate

®

_instrument

5 channels

for parallel tests

easy to use

Windows XP

based software

automatic analysis

and documentation

duplicate sensor for

internal quality control

electronic

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► _applications

Multiplate is already one of

the most widely used platelet

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put the test cell into the measuring position

attach the sensor cable 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}

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► _Multiplate

®

_reagents

comprehensive

line of dedicated

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TRAP platelet ac tivation GpIIb/IIIa r eceptor exposure degranulat ion ADP Arachidonic Acid Collagen ArA1 COX TXA₂ TXA2 COLtest ASPItest TRAPtest ADPtest ADPtest PGE1ADPtest HS (ADP + PGE1) activation inhibition GpIIb/IIIa antagonists: Reopro ® (abciximab) Aggrastat ® (Tirofiban) Integrillin ® (Eptifibatid) Aspirin ® NSAID Clopidogrel Prasugrel Cangrelor 1_{release of} arachidonic acid

► _{Multiplate tests 1/2}

rest ing plat elet activa tedpla telet

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► _{Multiplate tests 2/2}

mild vWD Bernard-Soulier

syndrome, severe vWD, aspirin

Ristocetin: vWF dependent platelet activation via the GpIb receptor RISTOtest

clopidogrel, vWF

aspirin, IIb/IIIa antagonists collagen: collagen activates platelet and

triggers a release of arachidonic acid from the platelet membrane, which is converted to TXA2 by the Cyclooxygenase

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

TRAP-6 (thrombin receptor activating

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

TRAPtest

aspirin, vWF clopidogrel, IIb/IIIa

antagonists ADP + prostaglandin E1 (Prostaglandin is

a natural inhibitor and enhances the sensitivity of the assay for clopidogrel) ADPtest HS

aspirin, vWF clopidogrel, IIb/IIIa

antagonists ADP: binds onto platelet ADP receptors

ADPtest

clopidogrel, vWF

aspirin, IIb/IIIa antagonists arachidonic acid: is converted to TXA2 by

platelet-own cyclooxygenase ASPItest not sensitive for sensitivity activation test

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► _{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%).

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► _{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.

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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

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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 U 139 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

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A.B. *1971

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university

clinic

Frankurt / Main

Hannover

medical school

Essen university

clinic

► _{near-patient application}

of Multiplate

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► _{Multiplate in interventional}

cardiology / neuroradiology

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Aggrastat Aggrastat

2 patients before and

after aggrastat loading

dose

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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.

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clopidogrel

2x75mg

clopidogrel

75mg

Non-response

Response

► _{increase of clopidogrel dose}

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Stent thrombosis

0 6 min AU

Clopidogrel 150mg/d

AU 0 6 min

Prasugrel 10mg/d

F. Krötz, Kardiologie, Klinikum der Universität München

► _{change from clopidogrel to prasugrel}

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cartridge-based platelet 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 ®

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