Multiplate® analysis takes place in a single-use test cell which incorporates dual sensors and a teflon-coated stirring magnet.  A key innovation of Multiplate® is the use of the Multiple Electrode Aggregometry (MEA) technology.

The principle of Multiplate® analysis is based on the fact that platelets become sticky upon activation and adhere and aggregate onto the metal sensor wires in the Multiplate® test cell. The test cell has a pipetting inlet and a cup portion with the sensor wires which extend into the blood sample. The sensor wires are made of highly conductive copper which are silver-coated. The test cell is connected to the instrument using a sensor cable and the electrical resistance between the sensor wires is recorded during the test. When activated, platelets adhere to the sensor wires and the electrical resistance between the wires increases.

  • Whole blood, no sample processing
  • Small sample volume (300 µl)
  • Mini test cells use 175 µl of whole blood; smallest volume requirement for aggregometry testing. Ideal for pediatric platelet research or animal platelet studies.
  • Up to 30 tests per hour
  • Interactive electronic pipetting
  • 5 independent channels for parallel testing
  • Specific and sensitive reagents
  • Standardized test procedures
  • Open system for research applications
  • Windows-based software
  • New tests can be defined in the system software
  • 1 × Multiplate® 5.0 Analyzer (MP0010)
  • 1 × Multiplate® Electronic Pipette (MP0030)
  • 1 × Multiplate® Pipette Tips (MP0040)
  • 1 × Multiplate® Sensor Cables (MP0360)
  • 1 × Multiplate® Test Cells (MP0027)
  • 1 × Multiplate® Reagent Rack (MP0370)
  • 1 × Multiplate® Keyboard (MP0441-EU)
  • 1 × Multiplate® Mouse (MP0032)
  • 1 × LCD monitor with hospital grade power cord (MP-MONITOR)
  • 1 × Printer (PRINTER)
  • 1 × Multiplate® Manual

Multiplate platelet function analyzer reagentsThe following reagents are available for Multiplate®


Platelet Function Testing Assay Reagents

The Multiplate® research analyzer records platelet aggregation at approximately 0.5 second intervals. The increase in impedance by the attachment of platelets onto the Multiplate® sensors is transformed to aggregation units (AU) and plotted against time.

Three parameters are calculated when performing research; Aggregation, AUC, and velocity. The most important parameter is the Area Under the aggregation Curve (AUC). AUC is recorded as Units or U. It is affected by the total height of the aggregation curve as well as by its slope and is best suited to express the overall platelet activity. The Aggregation (in AU) is the maximum height of the curve during the measurement period and the Velocity (in AU/min) is the maximum slope of the curve.

The output data calculated by the software is the mean value of the two independent sensors in the test cell. The correlation coefficient of the individual measurements is determined and the analysis is accepted when the correlation coefficient is greater than 0.98. Additionally, the difference of each curve from the mean curve (DIF) is calculated and is accepted when the difference is less than 20%.

[Note: An earlier version of the software expressed AUC as AU*min. The y-axis is aggregation in aggregation units (AU) and the x-axis is the time in minutes. To convert to current terminology, 10 AU*min = 1U.

Comparison of platelet aggregation using light transmission and multiple electrode aggregometry in Glanzmann thrombasthenia.
Awidi A, Maqablah A, Dweik M, Bsoul N, Abu-Khader A.
Platelets. Aug 2009; 20(5): 297-301.

Assessment of platelet function on whole blood by multiple electrode aggregometry in high-risk patients with coronary artery disease receiving antiplatelet therapy.
Paniccia R, Antonucci E, Maggini N, Romano E, Gori AM, Marcucci R, Prisco D, Abbate R.
Am J Clin Pathol. 2009; 131: 834-842.

Inhibition of platelet function with clopidogrel, as measured with a novel whole blood impedance aggregometer in horses.
Roscher KA, Failing K, Moritz A.
The Vet J. 2015; 203: 332-336.

Multiple electrode aggregometry: A new device to measure platelet aggregation in whole blood.
Tóth O, Calatzis A, Penz S, Losonczy H, Siess W.
Thromb Haemost. 2006; 96: 781-788.

How extensively has Multiplate® been used?
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.

What is routine maintenance/preventative maintenance?
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.
How is the data saved?
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.
What variables can be changed in 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.

What is the standard assay?
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.)
How are platelets activated?
Platelets are activated by agonists that are specific to individual receptors of the platelet. With Multiplate, the standard agonists are ADParachidonic 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.

Describe the training for Multiplate®.
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.
What are the nominal agonist concentrations of the Multiplate® assay?
TESTAGONISTREAGENT CONCENTRATION[AGONIST] in TEST CELL
ADPtestADP200 µM6.5 µM
ADPtest HSADP + PGE1200 µM + 300 nM6.25 µM + 9.4 nM
ASPItestarachidonic aid15 mM0.5 mM
COLtestcollagen100 µg/mL3.2 µg/mL
TRAPtestTRAP-6 peptide1000 µM32 µM
RISTOtestristocetin10 mg/mL
RISTOlow0.2 mg/mL
RISTOhigh0.77mg/mL
Controls
ASA controlacetylsalicylic acid30 mg/mL1 mg/mL
GPIIb/IIIa Antagonistsynthetic inhibitor50 µg/mL1.6 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.

How is standardization accomplished with the Multiplate® assay?
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.
What anticoagulants can be used with Multiplate® testing?
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.
What is Multiplate®?
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.