Advancing precision diagnostics in coagulation: introduction and analytical validation of a multiplex mass spectrometry–based test for 5 coagulation factors

BACKGROUND: Circulating levels and functionality of coagulation- and fibrinolysis-related proteins are critical in bleeding and thrombosis. Traditional assays lack multiplexing and adequate standardization and require substantial plasma volumes. Quantitative protein mass spectrometry (QPMS) enables multiplexed protein quantitation, addressing clinical and research gaps. OBJECTIVES: This study aimed to develop and validate a QPMS test for multiplexed quantitation of 5 coagulation proteins. METHODS: A QPMS test was developed to measure plasma fibrinogen (FBG), von Willebrand factor (VWF), factor [F]VIII, FIX, and FXI. Three tryptic peptides were chosen as representatives for each protein. Stable isotope-labeled internal standards were synthesized for precise quantitation. Analytical validation assessed precision, linearity, specificity, stability, carryover, reference intervals, method comparison, and matrix effects. RESULTS: Representative peptides for protein quantitation and identity confirmation were selected. Candidate peptides for FBG, VWF, FIX, and FXI produced sufficient signals in citrated and EDTA plasma, whereas FVIII candidate peptides did not. Validation (excluding FVIII) using plasmas from citrated healthy subjects and patients demonstrated median within-laboratory variations of 3.4% to 6.7% and a linear relationship (r = 0.990-0.998). Signals remained stable after 7 freeze/thaw cycles. A high level of agreement was shown with traditional assays (FBG: r = 0.991; VWF: r = 0.970; FIX: r = 0.926; FXI: r = 0.871). Robust relative quantitation of target proteins across different anticoagulant matrices was observed. CONCLUSION: We developed and validated a QPMS test for multiplexed quantitation of FBG, VWF, FIX, and FXI. Enrichment is required for low-abundant FVIII. This study indicates analytical feasibility for the use of QPMS in diagnostics and justifies development of 1 QPMS test encompassing all coagulation- and fibrinolysis-related proteins.