Antithrombin diagnostics by mass spectrometry: Development and analytical validation of a next-generation test

Area of Science:

• Biochemistry
• Molecular Diagnostics
• Proteomics

Background:

• Antithrombin deficiency is a rare but severe disorder associated with a high risk of thrombosis.
• Current diagnostic methods rely on activity tests, offering limited information on diverse antithrombin proteoforms.
• Mass spectrometry enables precision diagnostics through molecular characterization of proteins like antithrombin.

Purpose of the Study:

• To develop and validate a mass spectrometry-based assay for the molecular characterization of antithrombin.
• To establish a novel diagnostic tool for a more precise understanding of antithrombin deficiency.

Main Methods:

• Analytical validation of the mass spectrometry test, including imprecision, linearity, stability, and specificity.
• Explorative comparison with a commercial antithrombin activity test.
• Utilized liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometry.

Main Results:

• The validated test met predefined analytical performance specifications.
• Demonstrated a mean within-laboratory imprecision of 5.9% and linearity between 0.08 and 2.58 μmol/L.
• Showed a strong correlation (Pearson r = 0.88) with a commercial activity test for suspected quantitative deficiencies.

Conclusions:

• The validated mass spectrometry test provides molecular diagnosis for antithrombin deficiency.
• This assay can clarify ambiguous activity results and atypical clinical presentations.
• Future clinical studies aim to leverage this test for improved risk stratification and personalized medicine in thrombosis management.