Platelet count and mean platelet volume distribution profile could be determinate by modern haematology analyzers. Normal distribution of platelet volume is between 1.7 and 25.5 μm3 with a mean value of 6 μm3. An increased platelet volume is a prognostic factor for major acute cardiac events after acute myocardial infarction. In vivo bleeding time (Ivy and Mielke method) represents the time between the moment of a skin incision and the stop of bleeding. This test globally evaluates platetelet function, platelet-vascular wall interaction and vascular integrity and it is prolonged in case of dysfunction or damage of these components. Bleeding
detection system, measures changes in light transmission automatically and thus platelet-induced aggregation. The method is useful for quick detection of congenital or acquired platelet dysfunction, and for the evaluation of anti-platelet drugs efficacy: aspirin, clopidogrel and other thienopiridines, GPIIb/IIIa receptor antagonists in patients with acute coronary syndrome treated with platelet antagonists. TxB2 can be mea-sured in plasma to evaluate cyclooxygenase platelet activation, while its metabolites can be measured in urine. This in an indirect, not cost effective, not standardised method and can't be used for monitoring aspirin and other nonsteroidal anti-inflamatory drugs treatment. Flow-cytometric determination of vasodilator-stimulated phosphoprotein (VASP) phosphorylation is dependent on the activation level of the platelet P2Y12 receptor, which is targeted by clopidogrel. Vasodilator-stimulated phosphoprotein (VASP) is an intra-platelet actin regulatory protein. When compared to other methods, flow-cytometry analyses specific changes in size, granularity and surface antigen expression on individual platelets. It can evaluate platelet receptors status using monoclonal antibody against GPIIb/IIIa, CD 41 (integrin alpha 2b), PAC-1 (procaspase-activating compound 1) LIBS (ligand-induced binding sites) fibrinogen-RIBS (receptor-induced binding sites). Some limitations of the flow-cytometry assay for platelet function were described: Light transmission aggregometry -LTA-(Born Aggregometry) is a method based on the variation of the optical density of a particle suspension depending on the particle number and size. Light transmission aggregometry after ADP addition to the platelet rich plasma (PRP) was considered as the current gold standard to asses platelet function in response to clopidogrel. The technique is labor-intensive and restricted to specialised laboratories and has several disadvantages including sample preparation. The assay is time consuming, requires training to a high level of technichal proficiency; the methods for aggregation assay vary among laboratories, depending on the selection of agonists, agonist concentration, platelet concentration, time between sampling and analysis, centrifugation speed in preparing PRP with threefold difference in centrifugation speed (1000 rpm, 10 minutes when Chronolog Analyser is used, 720 g, 2 min for Bio/Data PAP4 Analyser and 200 g, 10 min for APACT Analyser). The assay can't be performed at the bedside and the blood samples must be quickly sent to an onsite laboratory. Other disadvantage is the fact that platelet functions are not similar in vitro and in vivo. All this data demonstrate that light transmission aggregometry is a time prolongation appears when platelet count is < 100000/mm3 or when platelet functions are inhibited (anti-platelet drugs, uraemia, liver dysfunction). Although bleeding time evaluates primary haemostasis, fibrinogen and plasma coagulation factors (V, VII, VIII, and IX) deficiency can affect the results. and for the diagnosis of congenital or acquired platelet dysfunction: Glanzmann-Naegeli thrombastenia, Bernard-Soulier syndrome, von Willebrand-Jürgens disease, storage and release defects, cyclooxigenase deficiency. In vitro bleeding time (Kratzer and Born method) can be determinated with Platelet Function Assay-100 (PFA-100), a global test for platelet function and vWF testing. Normal value of this test is 1-2 minutes and can be modiffied by GPIb receptor antagonists, GPIIb/IIIa receptor antagonists, von Willebrand factor (vWF) antagonists, aspirin, clopidogrel. In vitro bleeding time has a higher sensitivity when compared to in vivo bleeding time. When testing aspirin resistance and clopidogrel induced platelet inhibition, PFA-100 is less specific than aggregation based methods. Rapid Platelet Function Assay - RPFA - (Accumetrics® Verify now®) is a cartridge based, fully automated platelet function assay, with cartridges for thrombin receptor activated peptide (TRAP), aspirin and P2Y12 - the most important adenosine diphosphate (ADP) receptor on platelet surface inhibited by clopidogrel. RPFA is a turbidimetric-based optical method with low standardisation posibilities. The Multiplate Analyser is a multiple platelet function analyser and the method is usually named multiple electrode platelet aggregometry (MEA). This method functions with 5 independent channels, integrated computer, Windows XP based software, automatic analysis and documentation, electronic pipetting available and can detect the effects of aspirin, clopidogrel, GP IIb/IIIa antagonists and sensitivity for direct ADP receptor antagonists. The areas under the aggregation curve detected by each channel are compared, and if the difference is higher than 20% (vs. the mean curve) the measurement has to be repeated; the duplicate sensor serves as an internal control. The main application of the Multiplate system using the different test procedures is the monitoring of platelet function inhibitors. Several tests with different sensitivity towards the various anti-platelet agents are available. All Multiplate tests are affected by a blockade of the GPIIb/IIIa receptor. This shows that the binding of platelets to the metal sensors is dependent on the GPIIb/IIIa receptor.. The correlation coefficient (cc) between the values of the 2 individual curves is determined. The analysis is accepted when the cc is at least 0.98. The difference from the mean curve (DIF) is calculated based on the AUC values of the 2 individual measured curves. Arachidonic acid alone is not a platelet agonist. Therefore the platelet activation in ASPItest allows a very sensitive and specific detection of aspirin action. Arachidonic acid is the physiological substrate of the platelet COX. Aspirin and other nonsteroidal antiinflammatory drugs (NSAID) can block the platelet COX, resulting in a reduced aggregation in ASPItest. However, the potency of Aspirin is higher compared to NSAID. ASPItest can also be reduced in case of administration of other antiplatelets antagonists, platelet disorders, e.g. in hematological disorders, thrombocytopenia,. Higher ASPItest values in patients treated with aspirin demonstrate aspirin resistance, a normal response to aspirin therapy is with aggregation values below the cut-off (30 U in patients treated with aspirin). By definition, aspirin therapy should block arachidonic acid induced aggregation. Potential strategies to treat an aspirin lack of response would be either an increase of the aspirin dose (most likely treating aspirin non-response because of reduced aspirin absorption), an increase in frequency of aspirin administration (in case of suspected elevated turnover of platelets in the body), or switching the medication to a different drug (e.g. clopidogrel). ADPtest - Adenosine diphosphate test-platelets are activated by ADP, which triggers several receptors on the platelet surface. Clopidogrel and related drugs block the P2Y12 ADP receptor. ADPtest can also be reduced when other drugs interfering with platelet aggregation are taken, or in case of thrombocytopenia, platelet or other hematological disorders. An ingestion of aspirin leads typically to no or only a minor inhibition of aggregation in ADPtest. However some patients may also have a reduced platelet aggregation, due to other drugs interfering with platelet activity or due to comorbidities. Higher ADPtest values in patients treated with clopidogrel show an incomplete or no platelet inhibition and a normal response determine aggregation values below the cut-off (50 U in patients treated with clopidogrel). Patients non-responsiveness to clopidogrel could be treat with an increase of the clopidogrel dose, an increase in frequency of clopidogrel administration or switching the medication to aspirin or Aggrastat for short-term use. ADPtest HS-adenosine diphosphate test high sensitivity - In ADPtest under clopidogrel therapy an incomplete blockade of aggregation is often seen. This may be due to the fact that ADP not only triggers the P2Y12 ADP receptor on the platelet surface (i.e. the receptor that is blocked by clopidogrel), but also other ADP receptors (which are not affected by clopidogrel). A combination of ADP and a physiological platelet inhibitor (prostaglandin E1 = PGE1) can be more sensitive for the detection of the action of clopidogrel than the use of ADP alone. The binding of ADP to the P2Y12 receptor reduces the level of cAMP in the platelet which enhances the release of calcium from endogenous sources. The increase of intracellular calcium then leads to the activation and aggregation of the platelet. PGE1 reduces the mobilisation of calcium and thus inhibits platelet aggregation. As clopidogrel also reduces the platelet activation by ADP, clopidogrel and PGE1 act synergistically. Aggregation values in patients with normal response to clopidogrel are below the cut-off (25 U in patients treated with clopidogrel). COLtest - Collagen test - leads to platelet activation via the platelet collagen receptor. For suitable platelet activation endogenous arachidonic acid is released from the platelet phospholipids and is transformed to the platelet agonist thromboxane A2 by cyclooxygenase. Cyclooxygenase is blocked by aspirin, and therefore COLtest is sensitive to aspirin action. However this mechanism also explains why COLtest is less specific towards the action of aspirin compared to ASPItest. …platelet are activated through endogenous TXA2 and other TXA2 independent mechanisms. RISTOtest - Ristocetin test - Ristocetin is an antimicrobial substance which foms complexes with von Willebrand factor (vWF). In this complex vWF changes its conformation in a way that allows it to bind to platelets. Ristocetin is used on Multiplate in 2 concentrations: RISTOhigh: 0.77 mg/ml final concentration RISTOlow: 0.2 mg /ml final concentration. This allows the detection of samples with enhanced tendency for Ristocetin induced aggregation, especially von Willebrand disease (vWD) type IIB and samples with an absent or severely reduced response to Ristocetin (Bernard Soulier-Syndrome, vWD Type III, severe vWD Type I or II). Other disadvantages are represented by need of high sample stability, a long time for results availability, high costs of flow cytometer, request of experienced personel: COX inhibitors (aspirin, NSAID), ADP receptor antagonists (Clopidogrel, Prasugrel, Cangrelor) and GP IIb/IIIa antagonists (Abciximab, Tirofiban, Integrilin), provides a wide range of aggregation tests (ASPItest, ADPtest, ADPtest HS, TRAPtest, COLtest, RISTOtest), it is standardised and cost effective in comparisson with previously used methods.
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