Abstract: Objective To evaluate the susceptibility of alanine aminotransferase (ALT) and enzyme-linked immunosorbent assay (ELISA) employed in blood screening laboratories for hemolysis and lipemia samples, and to establish assay-specific interference thresholds for these variables. Methods Hemolysis samples with gradient hemoglobin (Hb) concentrations (0, 1, 2, 4, 5, 7.5 and 10 g/L) and lipemia samples with gradient triglyceride (TG) concentrations (0, 0.63, 1.27, 2.54, 5.08 and 10.15 mmol/L) were prepared. Interference effects were evaluated using three methods: gradient dilution linear regression, pre- and post-centrifugation bias analysis for ALT, (an absolute bias of ≤7.5% considered acceptable), and reactivity consistency testing for ELISA(maintaining an original reactivity/non-reactivity status). Based on these results, visual color charts for hemolysis and lipemia were developed. Results ALT demonstrated acceptable performance up to the highest tested triglyceride (TG) level (10.15 mmol/L), as evidenced by a linear regression slope of b=1, intercept a≈0, and absolute bias≤7.5%. ALT was also robust against hemolysis up to Hb=4 g/L (absolute bias≤7.5%). Among ELISA items, anti-HCV (sandwich method) and P24 antigen detected by HIV Ag/Ab reagent (4th generation) showed false negatives when TG=5.08 mmol/L. 3rd-generation and 4th-generation HIV Ag/Ab antibodies testing showed false negatives when Hb 4 g/L, while HBsAg testing showed false positives when Hb≥5 g/L. Considering these results of all items, the maximum acceptable interference thresholds were established as Hb 2 g/L and TG 2.54 mmol/L. Conclusion Hemolysis and lipemia significantly interfere with blood screening assay. Establishing unified, clinically relevant interference thresholds is essential to ensure result reliability and transfusion safety.

Key words: Hemolysis, Lipemia, Blood screening, Detection threshold