Multiplate® has been available since 2006 and is for Research Use Only in the US and Canada.
The instrument is used for research to assess the effect of antiplatelet drugs — aspirin, thienopyridines, dual antiplatelet therapy, PAR-1 inhibitors and to assess congenital platelet dysfunction. There are over 500 publications to date describing the methodology and its use in platelet testing.
The manufacturer recommends the sensor cables be replaced periodically. An annual service is available to check the system which includes temperature checks, stirring checks, electronic and liquid control testing. The electronic pipette is calibrated by the pipette manufacturer minimally on an annual basis. The Multiplate® analyzer does not require user calibration or adjustments.
The data is automatically saved as a .png file of the assay output (picture) and also as the raw data in aggregation units. The user can export the data via a flash drive and calculate aggregation for any time period or time segment of interest. Data is available as the individual assay or as the average of the assay.
With the preincubation step there is an opportunity to add three reagents and at the aggregation step one can add three reagents. Volume can be changed, temperature can be changed to a limited extent, and aggregation time can be measured from 3 to 20 minutes.
Currently a mini test cell is available which uses 0.175 mL diluent/0.175 mL whole blood/0.012 mL agonist to achieve the same concentrations as the standard test cell. The mini test cell can be used for small animal studies or situations where volume is critical. But, here one sees about 25% less aggregation as compared to the standard test cell.
The software-guided pipette prompts the user to add diluent (0.3 mL, 0.9% NaCl or 0.9% NaCl/3 mM CaCl2, agonist specific), whole blood (0.3 mL), and then agonist (0.02 mL) in the nominal three-step assay. The diluent/whole blood is incubated at 37° C for 3 minutes with stirring and aggregation, also with stirring, is measured for 6 minutes at 37° C in the standard assay for a total assay time of 9 minutes. (Note that each test cell comes with a PTFE stir bar.)
Platelets are activated by agonists that are specific to individual receptors of the platelet. With Multiplate, the standard agonists are ADP, arachidonic acid (AA), collagen, and TRAP (thrombin receptor activating peptide of 6 amino acids). These agonists bind to their specific receptors; P2Y1/P2Y12, TXA2 receptor after AA conversion by COX-1 to TXA2, GPVI, and PAR-1.
Ristocetin is also used as an agonist, however this produces platelet agglutination rather than aggregation by binding to GPIb/VWF (von Willebrand factor).
The user can test other agonists (e.g., PAR-4) and also evaluate the effect of antagonists on platelet function.
The Multiplate® instrument is very intuitive to use. Instrument setup takes about 2 hours and is performed by a trained individual of DiaPharma. Training include testing of a normal individual, data interpretation, electronic pipette use, how to create a pipette protocol, reagent preparation/use/storage, review of software features, and review of all maintenance activities. Generally a user can perform an assay after 30 minutes of instruction. Review of all instruments features typically requires 3 to 4 hours.
|TEST||AGONIST||REAGENT CONCENTRATION||[AGONIST] in TEST CELL|
|ADPtest||ADP||200 µM||6.5 µM|
|ADPtest HS||ADP + PGE1||200 µM + 300 nM||6.25 µM + 9.4 nM|
|ASPItest||arachidonic aid||15 mM||0.5 mM|
|COLtest||collagen||100 µg/mL||3.2 µg/mL|
|TRAPtest||TRAP-6 peptide||1000 µM||32 µM|
|ASA control||acetylsalicylic acid||30 mg/mL||1 mg/mL|
|GPIIb/IIIa Antagonist||synthetic inhibitor||50 µg/mL||1.6 µg/mL|
The calculation of agonist concentration in the test cell uses whole blood (WB) volume without consideration that approximately half of WB is red cell volume. Values derived from Multiplate product literature.
The test cell has a silver electrode of standardized length/diameter, the nominal assay is performed with defined and controlled time/volume/temperature, and the assay is done with defined agonist concentrations. Testing of a normal individual is not required with each assay.
The instrument manufacturer has provided reference ranges for three anticoagulants; lithium heparin (typically a green top blood collection tube), 3.2 % citrate (typically a blue top blood collection tube), and an RUO hirudin blood collection tube (3 mL) available from the instrument manufacturer. With the hirudin whole blood, the hirudin concentration in whole blood is >15 ug/mL. Note that the ACD blood collection tube (typically a yellow top blood collection tube) is not suitable for platelet function testing as low pH inhibits platelet function. Other anticoagulants require user validation for their suitability.
Multiplate® is a 5 channel instrument that measures the platelet aggregation of whole blood by impedance. Each disposable test cell has 2 independent electrode circuits. The instrument software measures the attachment of agonist-activated platelets to the electrode by the change in the electrical impedance at 0.57 second intervals. The software calculates the average of the two circuits for the aggregation measurement period. The data, after transformation, is in Units (U). Normal values are approximately between 40 to 125 Units, but the normal range of platelet function varies with the anticoagulant of the whole blood and with the agonist used for platelet activation.