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div>5 and 100?IU/mL). Although the performance of this assay is easier than washed platelets assays, its sensitivity was shown to vary largely depend on the selected donor and to be inferior to SRA . Most commonly, the heparin-induced platelet activation (HIPA) assay and the SRA [26, 27] are used. Both assays employ washed platelets and detect their activation by heparin-dependent antibodies by visually observing formation of platelet aggregates or release http://www.selleckchem.com/products/erastin.html of serotonin, respectively, and have similar operating characteristics. In the HIPA assay (Figure?2a), washed platelets from four healthy unselected donors are used. Washed platelets are incubated with the patient's serum in the presence of buffer (negative control), therapeutic heparin (0.2 anti-FXa/mL, reviparin), or supratherapeutic click here heparin (100?IU/mL, UFH) in a round bottom microtiter plate. Magnetic stirrers are used as source of shear force, and formation of platelet aggregates is visually determined every 5?min. The test is considered positive if there is platelet aggregation in therapeutic heparin concentrations with the platelet suspensions of at least two of four donors, but not in supratherapeutic heparin concentration, within 30?min. Test results in HIPA are quantitated depending on the lag time to platelet activation. In the SRA (Figure?2b), platelets obtained from a donor previously shown to be reactive to a panel of anti-PF4/heparin antibodies are used. The donor PRP is incubated with 14C-serotonin. Platelets take up the radiolabeled serotonin and store it in their dense granules. Platelets are then washed and incubated with patient's PF06463922 serum and heparin in flat-bottomed microtiter wells in duplicate on a plate shaker. After incubation for 60?min and centrifugation, supernatants of each reaction mixture are collected and their radioactivity is measured. Test results are expressed as percentage of serotonin release compared to a maximum after detergent-induced platelet lysis (100%). The test is considered positive if there is >20% release at therapeutic heparin levels and <20% release at supratherapeutic heparin levels. Different technical issues should be taken into consideration when both assays, HIPA and SRA, are performed: the addition of hirudin to quench all effects of residual thrombin in patients sample and the order of pipetting: (i) heat-inactivated serum, (ii) platelets, (iii) buffer, or LMWH. Heparin at high concentration and monoclonal antibody IV.3 should be added before the test platelets to disrupt PF4/heparin complexes and to block the Fc��IIa receptor, respectively, to prevent prompt activation of platelets in the presence of patient's serum which may result in false-positive results. Moreover, some differences exist between both assays. In SRA, UFH is used in different concentrations (0.1, 0.2, 0.3, and 100?IU/mL) and LMWH (enoxaparin) is used at 0.1?IU/mL.