Development and Validation of a Lectin-independent Liquid Chromatography–Tandem Mass Spectrometry Method for Serum Glycosylated Alpha-fetoprotein Analysis and Comparison with a Liquid-phase Binding Assay
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Background: Alpha-fetoprotein (AFP) and its isoform AFP-L3 are well-established serum biomarkers for hepatocellular carcinoma (HCC), a common malignancy and a leading cause of cancer-related mortality worldwide. Current methods for measuring these bio- markers are primarily lectin-based assays including the liquid-phase binding assay (LiBA) and liquid chromatography–tandem mass spectrometry (LC-MS/MS), both of which have limitations in diagnostic sensitivity and clinical utility for samples with low AFP concentra- tions. We aimed to develop a lectin-independent LC-MS/MS method for quantifying fu- cosylated AFP proteins (AFP-Fuc%). Methods: We conducted analytical validation, including method comparisons, over 2 months. The analytical sensitivity and diagnostic performance of this method were evalu- ated using 525 human serum samples—235 from HCC patients and 290 from non-HCC individuals—and compared with those of LiBA, which measured AFP-L3 levels. Results: The LC-MS/MS method demonstrated acceptable within-laboratory imprecision (CVs<17.1%) without detectable bias, carryover, or matrix effects. Our method exhibited a broader linear dynamic range (spanning five orders of magnitude) and 10-fold higher ana- lytical sensitivity than LiBA. The diagnostic performance of our method was significantly superior to that of LiBA, particularly in patients with low AFP concentrations (<7 ng/mL, P <0.001), with improved accuracy, sensitivity, and precision at a specificity of 96.2%. Conclusions: The validated LC-MS/MS method demonstrated robust analytical perfor- mance and superior diagnostic accuracy over LiBA for HCC diagnosis while avoiding the in- herent limitations of lectin-based assays. Our LC-MS/MS assay shows promise for early HCC detection and may contribute to enhanced patient care.
Abstract
Background: Alpha-fetoprotein (AFP) and its isoform AFP-L3 are well-established serum biomarkers for hepatocellular carcinoma (HCC), a common malignancy and a leading cause of cancer-related mortality worldwide. Current methods for measuring these bio- markers are primarily lectin-based assays including the liquid-phase binding assay (LiBA) and liquid chromatography–tandem mass spectrometry (LC-MS/MS), both of which have limitations in diagnostic sensitivity and clinical utility for samples with low AFP concentra- tions. We aimed to develop a lectin-independent LC-MS/MS method for quantifying fu- cosylated AFP proteins (AFP-Fuc%). Methods: We conducted analytical validation, including method comparisons, over 2 months. The analytical sensitivity and diagnostic performance of this method were evalu- ated using 525 human serum samples—235 from HCC patients and 290 from non-HCC individuals—and compared with those of LiBA, which measured AFP-L3 levels. Results: The LC-MS/MS method demonstrated acceptable within-laboratory imprecision (CVs<17.1%) without detectable bias, carryover, or matrix effects. Our method exhibited a broader linear dynamic range (spanning five orders of magnitude) and 10-fold higher ana- lytical sensitivity than LiBA. The diagnostic performance of our method was significantly superior to that of LiBA, particularly in patients with low AFP concentrations (<7 ng/mL, P <0.001), with improved accuracy, sensitivity, and precision at a specificity of 96.2%. Conclusions: The validated LC-MS/MS method demonstrated robust analytical perfor- mance and superior diagnostic accuracy over LiBA for HCC diagnosis while avoiding the in- herent limitations of lectin-based assays. Our LC-MS/MS assay shows promise for early HCC detection and may contribute to enhanced patient care.