BACKGROUND: Unexpected prolongation of first-line coagulation tests, including activated partial thromboplastin time (APTT), should trigger further work-up by performing mixing tests to elucidate the underlying cause, direct further specific testing and clarify their possible clinical impact. The aim of our study was to assess whether methodological diversity has any impact on the APTT mixing test results and their interpretation. MATERIAL AND METHODS: Two lyophilized plasma samples (case 1: heparin contamination [0.5 IU/mL]; case 2: factor VIII deficiency [0.13 IU/mL]) and their respective fictional clinical cases were sent to European laboratories for APTT measurement and performance of mixing tests. Participants were surveyed about the methodology (reagents, analytical platform, reference ranges), APTT results, mixing test conditions, their classification (normal, equivocal, prolonged) and categorization of the sample (factor deficiency, presence of inhibitor, anticoagulant, unknown). RESULTS: A total of 269 responses were included. For case 1, all participants reported a prolonged APTT, and 91% obtained no correction in the mixing test, without differences among reagents or analytical platforms. Only 15% of them selected the presence of an anticoagulant as the single cause for the prolongation. For case 2, 99% of participants reported a prolonged APTT, while some heterogeneity in the mixing test results was found. Eighty-six percent of participants selected factor deficiency as the cause for APTT prolongation. CONCLUSIONS: Most European laboratories obtained valid results for APTT and the subsequent mixing tests, despite using different methodologies. However, their classification could be improved. Therefore, more training and periodic evaluations are recommended to harmonize protocols and ensure proper result classification and categorization.