Irena Preložnik Zupan Tadej Pajič

Impedančna agregometrija

► Multiplate®

Analiza trombocitne funkcije v polni krvi na osnovi impedančne agregometrije

► 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 pipetting

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

► Multiplate® reagents

ADPtest: ADP induced platelet activation sensitive to clopidogrel, prasugrel …… ADPtest HS: Co-stimulation with ADP and PG E1, especially for the sensitive detection of clopidogrel in heparinized blood. ASPItest: Cyclooxygenase dependent aggregation (us ing arachidonic acid), sensitive to aspirin®, NSAIDs and other inhibitors of platelet cyclooxygenase. TRAPtest: Platelet stimulation via the thrombin receptor (using TRAP-6), sensitive to IIbIIIa receptor antagonists. COLtest: Collagen induced aggregation. RISTOtest: vWF and GpIb dependent aggregation (using ristocetin as agonist).

TRAP

ADP

Arachidonic Acid

Collagen

ArA1

COX

TXA2

TXA2

COLtest

ASPItest

TRAPtest

ADPtest

PGE1 ADPtest 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

► 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.

Parameters

► 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

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

A.B. *1971

very weak aggregation in all tests due to Glanzman Thrombasthenia

Aggrastat Aggrastat

2 patients before and after aggrastat loading

dose

aspirin 500 mg

i.v.

50 min after aspirin

application

aspirin 500 mg

i.v.

2 patients before and after aspirin 500 mg i.v.

clopidogrel 2x75mg

clopidogrel 75mg

Non- response

Response

► increase of clopidogrel dose

Stent thrombosis

0 6 min

AU

Clopidogrel 150mg/d

AU

0 6 min Prasugrel 10mg/d

► change from clopidogrel to prasugrel

university clinic

Frankurt / Main Hannover

medical school

Essen university clinic

► near-patient application of Multiplate

► Multiplate in interventional cardiology / neuroradiology

► applications

Multiplate is already one of the most widely used platelet

function tests

double-blind, placebo-controlled, randomized, three-period cross-over study in healthy volunteers designed to

compare the abilities of BT, LTA, and three point-of-care devices, Multiplate®, PFA100®, and VerifyNow® to quantitate the effects on platelet function of 3 days of treatment with aspirin, clopidogrel, or placebo.

From this study, we conclude that any one of the three simple-to-use point-of-care devices can reliably assess the treatment effect of ASA and CLP on platelet function in comparison with BT or LTA at the study population level

Whole blood aggregometry is dependent on all major cell lines in whole blood. Importantly, platelet aggregation is significantly associated with platelet count even within the normal range

št trombocitov

A ali V kri

napovedna vrednost

•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.

► Summary