Multiplate multiple electrode aggregometry (MEA) is a test of

platelet Platelets, also called thrombocytes (from Greek θρόμβος, "clot" and κύτος, "cell"), are a component of blood whose function (along with the coagulation factors) is to react to bleeding from blood vessel injury by clumping, thereby ini ...

function in

whole blood Whole blood (WB) is human blood from a standard blood donation. It is used in the treatment of massive bleeding, in exchange transfusion, and when people donate blood to themselves. One unit of whole blood (~517 mls) brings up hemoglobin lev ...

. The test can be used to diagnose platelet disorders, monitor antiplatelet therapy, and is also investigated as a potential predictor of transfusion requirements and bleeding risk in cardiac surgery.

Instrument

The Multiplate MEA Analyzer (Roche Diagnostics International Ltd) has 5 channels for simultaneous measurement of several samples or

agonists An agonist is a chemical that activates a receptor to produce a biological response. Receptors are cellular proteins whose activation causes the cell to modify what it is currently doing. In contrast, an antagonist blocks the action of the agon ...

. The instrument detects change in

electrical impedance In electrical engineering, impedance is the opposition to alternating current presented by the combined effect of resistance and reactance in a circuit. Quantitatively, the impedance of a two-terminal circuit element is the ratio of the comp ...

when platelets aggregate on metal

electrodes An electrode is an electrical conductor used to make contact with a nonmetallic part of a circuit (e.g. a semiconductor, an electrolyte, a vacuum or air). Electrodes are essential parts of batteries that can consist of a variety of materials de ...

in the test

cuvette A cuvette (French: cuvette = "little vessel") is a small tube-like container with straight sides and a circular or square cross section. It is sealed at one end, and made of a clear, transparent material such as plastic, glass, or fused quartz. ...

. Each cuvette contains two pairs of sensor electrodes, each of which measures the change in impedance. The duplicate sensors serve as an integrated quality control, and the analysis is accepted if the

correlation coefficient A correlation coefficient is a numerical measure of some type of correlation, meaning a statistical relationship between two variables. The variables may be two columns of a given data set of observations, often called a sample, or two components ...

of the measurements is greater than 0.98. The difference of each curve from the mean curve is also calculated, and a difference of less than 20% is accepted. The test cuvettes also contain a

teflon Polytetrafluoroethylene (PTFE) is a synthetic fluoropolymer of tetrafluoroethylene that has numerous applications. It is one of the best-known and widely applied PFAS. The commonly known brand name of PTFE-based composition is Teflon by Chemou ...

coated

magnetic Magnetism is the class of physical attributes that are mediated by a magnetic field, which refers to the capacity to induce attractive and repulsive phenomena in other entities. Electric currents and the magnetic moments of elementary particle ...

stirring bar. The increase in impedance as aggregation occurs is transformed into aggregation units (AU) and plotted against time on the computer screen.

Sample

The manufacturer recommends the use of

hirudin Hirudin is a naturally occurring peptide in the salivary glands of blood-sucking leeches (such as '' Hirudo medicinalis'') that has a blood anticoagulant property. This is fundamental for the leeches’ habit of feeding on blood, since it keeps ...

anticoagulant Anticoagulants, commonly known as blood thinners, are chemical substances that prevent or reduce coagulation of blood, prolonging the clotting time. Some of them occur naturally in blood-eating animals such as leeches and mosquitoes, where the ...

for samples to be tested, but studies have shown that

heparin Heparin, also known as unfractionated heparin (UFH), is a medication and naturally occurring glycosaminoglycan. Since heparins depend on the activity of antithrombin, they are considered anticoagulants. Specifically it is also used in the treatm ...

is a good alternative. 300 µL of blood is needed for each analysis, and is diluted with the same amount of saline. After pipetting blood and saline into the cuvette, the test is incubated for three minutes before the chosen agonist is added. The test is then started, and platelet aggregation is recorded at approximately 0.5 second intervals for six minutes. Three parameters are calculated: aggregation,

velocity Velocity is the directional speed of an object in motion as an indication of its rate of change in position as observed from a particular frame of reference and as measured by a particular standard of time (e.g. northbound). Velocity is a ...

and

area under the curve In mathematics, an integral assigns numbers to functions in a way that describes displacement, area, volume, and other concepts that arise by combining infinitesimal data. The process of finding integrals is called integration. Along with ...

(AUC). Aggregation (in AU) is the

maximum In mathematical analysis, the maxima and minima (the respective plurals of maximum and minimum) of a function, known collectively as extrema (the plural of extremum), are the largest and smallest value of the function, either within a given ran ...

height Height is measure of vertical distance, either vertical extent (how "tall" something or someone is) or vertical position (how "high" a point is). For example, "The height of that building is 50 m" or "The height of an airplane in-flight is abou ...

of the aggregation

curve In mathematics, a curve (also called a curved line in older texts) is an object similar to a line (geometry), line, but that does not have to be Linearity, straight. Intuitively, a curve may be thought of as the trace left by a moving point (ge ...

, and velocity (in AU/min) is the maximum

slope In mathematics, the slope or gradient of a line is a number that describes both the ''direction'' and the ''steepness'' of the line. Slope is often denoted by the letter ''m''; there is no clear answer to the question why the letter ''m'' is use ...

of the curve. AUC is the most important

parameter A parameter (), generally, is any characteristic that can help in defining or classifying a particular system (meaning an event, project, object, situation, etc.). That is, a parameter is an element of a system that is useful, or critical, when ...

. It is recorded in Units (U), and is affected by both the height and the slope of the aggregation curve, and is the best overall measure of platelet activity.

Multiplate Tests/Reagents

ADPtest

Adenosine diphosphate Adenosine diphosphate (ADP), also known as adenosine pyrophosphate (APP), is an important organic compound in metabolism and is essential to the flow of energy in living cells. ADP consists of three important structural components: a sugar backbon ...

(ADP) is a platelet agonist. When it is added to saline-diluted whole blood in the test cuvette, it stimulates the ADP

receptor Receptor may refer to: * Sensory receptor, in physiology, any structure which, on receiving environmental stimuli, produces an informative nerve impulse *Receptor (biochemistry), in biochemistry, a protein molecule that receives and responds to a ...

s on platelets, activating the platelets. The activation of the platelets leads to shape change and degranulation, and the released content of the granules further activates the platelets. Activation also induces a

conformational change In biochemistry, a conformational change is a change in the shape of a macromolecule, often induced by environmental factors. A macromolecule is usually flexible and dynamic. Its shape can change in response to changes in its environment or oth ...

in the

glycoprotein IIb/IIIa In medicine, glycoprotein IIb/IIIa (GPIIb/IIIa, also known as integrin αIIbβ3) is an integrin complex found on platelets. It is a receptor for fibrinogen and von Willebrand factor and aids platelet activation. The complex is formed via calcium ...

(GPIIb/IIIa) receptor, giving it high

affinity Affinity may refer to: Commerce, finance and law * Affinity (law), kinship by marriage * Affinity analysis, a market research and business management technique * Affinity Credit Union, a Saskatchewan-based credit union * Affinity Equity Par ...

for

fibrinogen Fibrinogen (factor I) is a glycoprotein complex, produced in the liver, that circulates in the blood of all vertebrates. During tissue and vascular injury, it is converted enzymatically by thrombin to fibrin and then to a fibrin-based blood clo ...

. Binding of fibrinogen to GPIIb/IIIa receptors leads to platelet-to-platelet bridges and results in platelet aggregation.

Antiplatelet drugs An antiplatelet drug (antiaggregant), also known as a platelet agglutination inhibitor or platelet aggregation inhibitor, is a member of a class of pharmaceuticals that decrease platelet aggregation and inhibit thrombus formation. They are effecti ...

clopidogrel Clopidogrel — sold under the brand name Plavix, among others — is an antiplatelet medication used to reduce the risk of heart disease and stroke in those at high risk. It is also used together with aspirin in heart attacks and following t ...

and

prasugrel Prasugrel, sold under the brand name Effient in the US, Australia and India, and Efient in the EU) is a medication used to prevent formation of blood clots. It is a platelet inhibitor and an irreversible antagonist of P2Y12 ADP receptors and is ...

irreversibly inhibit the ADP receptor

P2Y12 P2Y12 is a chemoreceptor for adenosine diphosphate (ADP) that belongs to the Gi class of a group of G protein-coupled (GPCR) purinergic receptors. This P2Y receptor family has several receptor subtypes with different pharmacological selec ...

, leading to a decreased ADP-induced platelet aggregation. Drugs that inhibit the GPIIb/IIIa receptor, e.g.

eptifibatide Eptifibatide (Integrilin, Millennium Pharmaceuticals, also co-promoted by Schering-Plough/Essex), is an antiplatelet drug of the glycoprotein IIb/IIIa inhibitor class. Eptifibatide is a cyclic heptapeptide derived from a disintegrin protein () ...

, can also reduce or eliminate the ADP-induced platelet response.

ASPItest

In the ASPItest

arachidonic acid Arachidonic acid (AA, sometimes ARA) is a polyunsaturated omega-6 fatty acid 20:4(ω-6), or 20:4(5,8,11,14). It is structurally related to the saturated arachidic acid found in cupuaçu butter. Its name derives from the New Latin word ''arachi ...

is added to the saline-diluted blood sample. Arachidonic acid is converted to

prostaglandin H2 Prostaglandin H2 is a type of prostaglandin and a precursor for many other biologically significant molecules. It is synthesized from arachidonic acid in a reaction catalyzed by a cyclooxygenase enzyme. The conversion from Arachidonic acid to P ...

(PGH2) by

cyclooxygenase-1 Cyclooxygenase 1 (COX-1), also known as prostaglandin G/H synthase 1, prostaglandin-endoperoxide synthase 1 or prostaglandin H2 synthase 1, is an enzyme that in humans is encoded by the ''PTGS1'' gene. In humans it is one of two cyclooxygenases. ...

(COX1), and PGH2 is then converted to

thromboxane A2 Thromboxane A2 (TXA2) is a type of thromboxane that is produced by activated platelets during hemostasis and has prothrombotic properties: it stimulates activation of new platelets as well as increases platelet aggregation. This is achieved by act ...

(TXA2) by

thromboxane synthase Thromboxane A synthase 1 (, platelet, cytochrome P450, family 5, subfamily A), also known as TBXAS1, is a cytochrome P450 enzyme that, in humans, is encoded by the ''TBXAS1'' gene. Function This gene encodes a member of the cytochrome P450 supe ...

. TXA2 increases platelet aggregation, promotes degranulation and stimulates platelet activation. Inhibition of COX1, as with

acetylsalicylic acid Aspirin, also known as acetylsalicylic acid (ASA), is a nonsteroidal anti-inflammatory drug (NSAID) used to reduce pain, fever, and/or inflammation, and as an antithrombotic. Specific inflammatory conditions which aspirin is used to treat inc ...

, and inhibition or absence of GPIIb/IIIa receptor, as seen in

Glanzmann's thrombasthenia Glanzmann's thrombasthenia is an abnormality of the platelets. It is an extremely rare coagulopathy (bleeding disorder due to a blood abnormality), in which the platelets contain defective or low levels of glycoprotein IIb/IIIa (GpIIb/IIIa), which ...

, will reduce platelet aggregation in response to arachidonic acid.

COLtest

Collagen Collagen () is the main structural protein in the extracellular matrix found in the body's various connective tissues. As the main component of connective tissue, it is the most abundant protein in mammals, making up from 25% to 35% of the whole ...

is added to the sample-saline mix, and binds to collagen-receptors on platelets. This leads to a release of arachidonic acid, which is converted to the potent platelet activator TXA2. COLtest is sensitive to inhibition of COX1 and GPIIb/IIIa and to

RISTOtest

Ristocetin Ristocetin is a glycopeptide antibiotic, obtained from ''Amycolatopsis lurida'', previously used to treat staphylococcal infections. It is no longer used clinically because it caused thrombocytopenia and platelet agglutination. It is now used so ...

forms complexes with

von Willebrand factor Von Willebrand factor (VWF) () is a blood glycoprotein involved in hemostasis, specifically, platelet adhesion. It is deficient and/or defective in von Willebrand disease and is involved in many other diseases, including thrombotic thrombocytopen ...

(vWF) that bind to the

glycoprotein Ib Glycoprotein Ib (GPIb), also known as CD42, is a component of the Glycoprotein Ib-IX-V Receptor Complex, GPIb-V-IX complex on platelets. The GPIb-V-IX complex binds von Willebrand factor, allowing platelet adhesion and platelet plug formation at si ...

(GP1b) receptors on platelets, causing platelet activation and aggregation. Reduced or absent aggregation in response to ristocetin can be caused by a lack of or reduction of vWF, as seen in

von Willebrand disease Von Willebrand disease (VWD) is the most common hereditary blood-clotting disorder in humans. An acquired form can sometimes result from other medical conditions. It arises from a deficiency in the quality or quantity of von Willebrand factor ( ...

(vWD), or absence or reduction in numbers of GP1b receptors, as in

Bernard–Soulier syndrome Bernard–Soulier syndrome (BSS) is a rare autosomal recessive bleeding disorder that is caused by a deficiency of the ''glycoprotein Ib-IX-V complex'' (GPIb-IX-V), the receptor for von Willebrand factor. The incidence of BSS is estimated to be l ...

(BSS). RISTOtest can be performed in two concentrations; RISTOhigh and RISTOlow. In RISTOhigh, a ristocetin concentration of 0,77 mg/ml is applied. This normally induces a strong platelet aggregation, and low or absent aggregation can be caused by the earlier mentioned conditions vWD and BSS. In RISTOlow, the ristocetin concentration is just 0,2 mg/ml, and at a level that does not normally induce a strong aggregation response. This test can detect if vWF shows a stronger than normal tendency to aggregate platelets, which can be seen in a subtype of vWD called vWDIIb.

TRAPtest

Thrombin receptor activating peptide-6 (TRAP-6) activates platelets through the

thrombin receptor There are three known thrombin receptors (ThrR), termed PAR1, PAR3 and PAR4 (PAR for protease-activated receptor). G-protein-coupled receptors that are responsible for the coagulation effects and responses of thrombin on cells are known as pro ...

protease activated receptor-1 (PAR-1). Binding of TRAP-6 to PAR-1 causes a conformational change in the GPIIb/IIIa receptors on platelets, giving them high affinity for fibrinogen. Fibrinogen then binds to the receptors,

crosslinking Cross-linking may refer to *Cross-link In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers ca ...

several platelets and causing aggregation. Aggregation in TRAPtest may be reduced or absent in

, where GPIIb/IIIa receptors are few or absent, or if the patient is taking a GPIIb/IIIa

antagonist An antagonist is a character in a story who is presented as the chief foe of the protagonist. Etymology The English word antagonist comes from the Greek ἀνταγωνιστής – ''antagonistēs'', "opponent, competitor, villain, enemy, riv ...

. Traptest has only minor sensitivity for inhibition of COX1 and ADP-receptors.

ASA Reagent

Adding ASA (

) reagent to a blood sample reduces the aggregation responses in ASPItest and COLtest. ASA irreversibly inhibits COX1 in platelets, thereby inhibiting the production of TXA2. The ASA Reagent is used as a quality control in the platelet function testing with Multiplate, allowing the assessment of abnormal platelet responses in ASPItest and COLtest.

GPIIb/IIIa Antagonist Reagent

This reagent is used as a quality control in Multiplate platelet function testing. The GPIIb/IIIa antagonist blocks the binding of fibrinogen to the GPIIb/IIIa receptors, preventing the formation of platelet-fibrinogen bonds and resulting in significantly reduced platelet aggregation in response to all agonists. The antagonist reagent is used together with TRAP-test, and allows assessment of a positive control.

Prostaglandin E1 Reagent

Prostaglandin E1 Prostaglandin E1 (PGE1), also known as alprostadil, is a naturally occurring prostaglandin which is used as a medication. In infants with congenital heart defects, it is delivered by intravenous infusion, slow injection into a vein to open the d ...

(PGE1) is a platelet inhibitor that causes an increase in

cyclic adenosine monophosphate Cyclic adenosine monophosphate (cAMP, cyclic AMP, or 3',5'-cyclic adenosine monophosphate) is a second messenger important in many biological processes. cAMP is a derivative of adenosine triphosphate (ATP) and used for intracellular signal transd ...

(cAMP) in platelets by stimulating

adenylyl cyclase Adenylate cyclase (EC 4.6.1.1, also commonly known as adenyl cyclase and adenylyl cyclase, abbreviated AC) is an enzyme with systematic name ATP diphosphate-lyase (cyclizing; 3′,5′-cyclic-AMP-forming). It catalyzes the following reaction: :A ...

activity. cAMP is an intracellular

signaling molecule In biology, cell signaling (cell signalling in British English) or cell communication is the ability of a cell to receive, process, and transmit signals with its environment and with itself. Cell signaling is a fundamental property of all cellula ...

, and the increase in cAMP inhibits

calcium Calcium is a chemical element with the symbol Ca and atomic number 20. As an alkaline earth metal, calcium is a reactive metal that forms a dark oxide-nitride layer when exposed to air. Its physical and chemical properties are most similar to ...

mobilization and platelet aggregation induced by activation of the ADP receptor P2Y1. Activation of the P2Y1 receptor initiates platelet aggregation in response to ADP. The P2Y1 receptor is required for ADP-induced platelet activation, but is not sufficient for a full platelet aggregation in response to ADP. cAMP inhibits platelet aggregation, and decreased amounts of cAMP in platelets lead to platelet aggregation. The PGE1 reagent is used together with the ADP test for two purposes: the assessment of ADP high sensitivity (HS) and of positive controls of the ADPtest. When a small amount of PGE1 (20 µl) is added to the ADPtest, it induces a moderat inhibition of aggregation in normal samples, but greatly increases the sensitivity of platelets to inhibition by clopidogrel. This is why the modified test is called "High Sensitivity". Adding 50 µl of PGE1 to the ADPtest leads to a strong inhibition of ADP induced aggregation, and is used for assessment of positive controls of the ADPtest.

References

{{reflist Medical tests Medical testing equipment