A targeted mass spectrometric assay for quantitative detection of fibrinogen in plasma.
Abstract
Fibrinogen is a key glycoprotein that plays a central role in clot formation, structure, and stability. Both quantitative fibrinogen deficiency and qualitative abnormalities can lead to bleeding or thrombotic disorders. Accurate quantification of fibrinogen antigen is essential for clinical evaluation; however, current immunoassays and clot-based surrogate methods are prone to interference and may lack traceability to international standards. To develop a targeted mass spectrometry-based assay for accurate quantification of fibrinogen antigen in plasma and to compare its performance with established functional and immunoturbidimetric assays. We developed a targeted liquid chromatography-tandem mass spectrometry-based assay using isotope-labeled peptides that accurately measures fibrinogen antigen concentration with high precision (coefficient of variation < 20%) across a broad linear dynamic range of 10.2 to 1307.5 mg/dL, enabling accurate detection of hypofibrinogenemia and hyperfibrinogenemia. Assay performance was evaluated in healthy donors (n = 47) and clinical specimens (n = 31). Fibrinogen antigen levels in normal donors followed a normal distribution (mean ± SD, 315 ± 42 mg/dL). The targeted assay, employing selected reaction monitoring, correlated strongly with immunoturbidimetry (Pearson's correlation coefficient [r] = .943) and the Clauss and prothrombin time-derived fibrinogen assays (r = .91 and r = .93, respectively). Stratification by clinical category confirmed elevated fibrinogen levels in thrombosis-related disorders, intermediate levels in controls, and low levels in liver disease and critical illness. The liquid chromatography-tandem mass spectrometry-based assay provides precise, accurate quantification of fibrinogen across a wide clinical range and aligns closely with existing methods while offering analytical specificity. With further clinical validation, it could be used as a diagnostic clinical test.
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