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A Practical Guide to Haemostasis


Platelet Function Testing:
VerifyNow Assays

Introduction

1. The VerifyNow-P2Y12 Assay/VerifyNow PRU Test
The VerifyNow-P2Y12 Assay/VerifyNow PRU Test is a rapid test that uses ADP to stimulate platelets in the presence of PGE1 [Prostaglandin E1] and which inhibits activation downstream of a second ADP receptor P2Y1 - making the assay more sensitive and specific for the activity of the P2Y12 receptor and of drugs that bind to the P2Y12 receptor. The assay system reagent is designed to specifically measure P2Y12 - mediated platelet aggregation.

There are two principal ADP receptors on the surface of the platelets:
a. P2Y1 P2Y1 is a G-coupled receptor that initiates ADP-induced platelet aggregation through stimulation of Phospholipase C and the Phosphatidylinositol-signaling pathway.
b. P2Y12 : P2Y12 is a G-coupled seven-transmembrane domain receptor, which mediates platelet activation by inhibiting the Adenylate Cyclase signalling pathway and decreasing intracellular cAMP levels.  The decrease in cAMP activates the GPIIb/IIIa receptor and platelet aggregation.
P2Y12 plays a central role in amplification and stabilisation of ADP-induced platelet aggregation.

2. VerifyNow Aspirin Assay
The methodology is very similar to the VerifyNow-P2Y12 Assay/VerifyNow PRU Test but Arachidonic Acid is incorporated to measure the response of the platelet to Aspirin.  In an individual on Aspirin, Aspirin irreversibly inhibits the COX-1, the enzyme that converts Arachidonic acid to Thromboxane A [TxA2] and which ultimately activates the GPIIb/IIIa receptor involved in platelet aggregation. In the presence of Aspirin the aggregation does not occur.

Principles & Methodology

The VerifyNow System is a turbidometric based optical detection system utilising microbeads and which measures platelet induced aggregation as an increase in light transmittance.


1. P2Y12 Assay System
a. The patient citrated whole blood sample is introduced into the assay system.
b. The assay consists of Fibrinogen-coated microbeads and the platelets are activated by the inclusion of ADP.
c. Activation of platelets via the ADP [20 μmol]  in the device activates the GPIIb-IIIa receptor and this then binds to the Fibrinogen-coated microbeads.  The device also contains PG E1 which reduces activation of the P2Y1 receptor and therefore makes the assay specific for the P2Y12 receptor.
e. Aggregation of the beads occurs and and this leads to a change in OD which is measured.

The VerifyNow P2Y12 assay reports results in P2Y12 Reaction Units (PRU).
Baseline platelet activity [termed BASE] in the absence of platelet P2Y12 receptor inhibition is achieved by using a modified Thrombin-receptor Activating Peptide [TRAP] in a reference channel. 

The results of the assay (TEST) are reported as:
i. P2Y12-Reaction-Units (PRU)
ii. Percent inhibition (% inhibition):  [BASE PRU - Post-Treatment PRU]/[Baseline PRU] × 100.

2. VerifyNow Aspirin Assay
The VerifyNow Aspirin Assay reports results in Aspirin Reaction Units (ARU).
The methodology for the assay is very similar to the VerifyNow-P2Y12 Assay/VerifyNow PRU Test but Arachidonic Acid is incorporated to measure the response of the platelet to Aspirin. 

Reference Ranges


1. VerifyNow-P2Y12 Assay:  .
  >180 - 376 PRU [P2Y12 Reaction Units]  - No drug present
  10-180 PRU [P2Y12 Reaction Units]  - Decreased platelet reactivity to P2Y12 inhibitor.

2. VerifyNow-Aspirin Assay
  ≥ 550 Aspirin Reaction Units [ARU] - No evidence of platelet dysfunction due to Aspirin.
  < 550 Aspirin Reaction Units [ARU] - Platelet dysfunction consistent with Aspirin - detected.