This assay showed better discrimination than two commercial immunoassays. from interlaboratory variability. This lack of standardization limits the evaluation and the convenience of practical assays in laboratories. In the present article, we review all the current activation endpoints, techniques and methodologies of practical assays developed for HIT analysis. [120]. Donor platelet reactivity may be tested in platelet aggregation assay with common platelet activators such as ADP, collagen, arachidonic acid or Capture [33,40,41,77]. Some laboratories proposed a positive IgG-specific anti-PF4/heparin EIA as a quality control to avoid a false-positive SRA statement as incongruous results may occur (i.e., positive SRA in combination with bad EIA and an atypical medical demonstration) [12,14,22,94,121,122]. 8. Additional Variations Donor platelets are incubated with patient serum/plasma and heparin in all practical assays. This preanalytical step may differ for the agitation pressure, the incubation time and the incubation heat among different practical assays or for the same practical assay. The incubation heat may vary from space heat [24,27,34,45] to 25C28 C [48,51] to 37 C [45,55]. To agitate, occasional gentle combining [24,48], low rate [27], agitation of 1000 rpm [29,34,42] or 1200 rpm [55] are used. The incubation period may vary from 15 min [41,42] to 20 min [55,56] to 30 min [45,48,51] to 45 min [34] for up to 60 min [22,27,45]. The percentage of donor platelets to the patient sample is usually 3.75:1 for washed platelet-based assays [22,27] and usually between 1:0.5 and 1:1 for PRP and whole blood-based assays [36,39,40,55]. To collect donor platelets, ACD, citrate or hirudin tubes are often used. ACD tubes are used for washed platelet-based assays [22,27,58]. Citrate tubes are commonly utilized for PRP or whole blood-based assays [19,39,42,44,55]. Hirudin tubes are often used preferably for HIMEA as it enhances assay level of sensitivity [39,89] by avoiding CalDAG-GEFII issues of calcium concentrations influencing platelet response (and also the problem of calcium depletion in under-filled citrate tubes [41]). Heparin tubes are avoided in order to prevent increase of final heparin concentration in the test. 9. Results Manifestation Results expression is definitely specific to the endpoint and the technology used. Functional assays using the release of radiolabeled or unradiolabeled serotonin as an endpoint (14C-SRA, EIA-SRA, HPLC-SRA) often express the results as a percentage of serotonin launch to account for inter- and intra-individual variability in platelet serotonin content material [25,27]. Manifestation of natural serotonin ideals may be used [24,25,28]. Platelet activation assays measuring ATP launch reported luminescence results in moles of ATP Indolelactic acid per amount of platelets [29]. HIPA results are indicated as the presence or absence of a visual platelet Indolelactic acid aggregation [34]. PAT results are indicated as the area under the aggregation curve [44] or Indolelactic acid as percentage of aggregation [40,123]. HIMEA results are most commonly indicated as the area under the aggregation curve [39,40,42,43]. The aggregation velocity and the lag-time may be also used [41,89]. Circulation cytometry experiments measuring the manifestation of platelet activation markers communicate results as a percentage of triggered platelets [46,48,49,50,124]. Circulation cytometry assays that detect generated PMPs may communicate results as the amount or the percentage of PMPs [51,58]. For the FcRIIa proteolysis assay, results are identified as the percentage of proteolysis [64]. Some studies express the final results as a percentage between results at the low heparin concentration and at the high heparin concentration [51,55,56,95] or like a percentage between results at the low heparin concentration and in the absence of heparin [61]. The percentage low/high heparin concentration takes into account the heparin dependency confirmation step in the final result manifestation but is not relevant to each practical assay as the result in Indolelactic acid the high heparin concentration may be zero in HIMEA for instance [41]. 10. Results Interpretation According to the results of the test condition and settings, several situations are possible (Table 1). Table 1 Different patterns with a combination of functional assay results (platelet response) at four test conditions (i.e., absence of added heparin, low concentration(s) and high concentration of heparin and monoclonal antibody IV.3). Potential causes of each pattern are provided. Negative and positive settings are not displayed in the table. Neg: bad, Pos: positive, Ab: antibody, IgG: immunoglobulin G, HLA:.