Novel insights into antithrombin deficiency enabled by mass spectrometry-based precision diagnostics

Area of Science:

• Biochemistry
• Proteomics
• Clinical Diagnostics

Background:

• Current antithrombin deficiency (ATD) diagnosis uses crude tests, leading to patient heterogeneity and overlooked thrombophilia phenotypes.
• Existing clinical pathways and evidence rely on these tests, resulting in generic treatment approaches.

Purpose of the Study:

• To develop and evaluate a mass spectrometry-based test for molecular characterization of antithrombin proteoforms in ATD patients.
• To address the heterogeneity of ATD by providing a more precise diagnostic tool.

Main Methods:

• Liquid chromatography coupled to multiple-reaction-monitoring mass spectrometry (LC-MRM-MS) was used to analyze antithrombin proteoforms in plasma.
• Plasma samples from 91 unrelated ATD patients and 41 patients with congenital disorders of glycosylation were analyzed.

Main Results:

• The LC-MRM-MS test achieved 100% diagnostic sensitivity for ATD, surpassing the 81.1% sensitivity of functional tests.
• It accurately differentiated between type I and type II ATD and identified variant proteoforms.
• For type II ATD, a subtype prone to misdiagnosis, LC-MRM-MS showed 100% identification versus 56.8% for functional tests.

Conclusions:

• Mass spectrometry-based antithrombin testing offers a platform for investigating ATD's molecular basis and clinical heterogeneity.
• This precision diagnostics approach can reduce diagnostic uncertainty and modernize ATD diagnostic and clinical pathways.