Evaluation of the acceptability of proposed hemolysis limits for Werfen ACL-TOP 750 CTS

INTRODUCTION: Hemolysis is a common preanalytical problem in coagulation testing. The Werfen ACL TOP 750CTS system detects hemolysis by measuring optical absorbance to estimate cell-free hemoglobin. This study assessed whether the manufacturer’s proposed hemolysis cut-offs are reliable for routine use. METHODS: Ten citrated whole blood samples were divided into four aliquots and subjected to increasing mechanical hemolysis by repeated aspirations through a 1-mL insulin syringe with a 25-gauge needle. Aliquots underwent 0, 4, 8, or 12 aspirations to generate a hemolysis gradient. Plasma was separated and tested for prothrombin time (PT), activated partial thromboplastin time (aPTT), antithrombin activity (AT), D-dimer (DD), and fibrinogen (Clauss method) on ACL TOP. Clinically significant differences were defined using reference change values (RCVs) and compared to manufacturer’s H-index thresholds (5.00 g/L for PT, APTT, AT and DD, 3.75 g/L for fibrinogen). RESULTS: Baseline hemolysis index (H-index) ranged between 0.28-1.04 g/L and increased proportionally with aspiration cycles. After 12 aspirations, 9 of 10 samples exceeded 9.00 g/L. Up to 8 aspirations (H-index between 60-282 mg/dL), all variations remained within the RCVs. After 12 aspirations, PT was measurable in 8/10 samples (variation -3.6% to 13.5%), aPTT in 7/10 (-13.8% to 11.5%), fibrinogen in 2/10 (-0.9% to 17.4%), DD in 6/10 (-3.3% to 169.0%), AT in 5/10 (-30.4% to -13.0%). DISCUSSION: The manufacturer’s hemolysis cut-offs for ACL TOP 750CTS are suitable for routine use. Hemolysis did not significantly affected routine coagulation assays except at extremely high cell-free hemoglobin concentrations, where the analyzer appropriately suppressed results.