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

Research and Practice in Thrombosis and Haemostasis, vol. 7, pp. 100079

Abstract

Background: Antithrombin deficiency is a rare but severe disorder leading to high risk of thrombosis. The current clinical care pathway relies on activity tests, which only provide overall functional information on the in vitro activity of antithrombin. However, antithrombin exists in many different forms, also known as proteoforms, with varying clinical phenotypes. Precision diagnostics, facilitated by mass spectrometry, provides a strategy to improve patient diagnostics by molecular characterization. Objectives: To develop and analytically validate a mass spectrometry-based test for molecular characterization of antithrombin. Methods: The test was analytically validated based on predefined analytical performance specifications. The validation covered imprecision, carryover, linearity, stability, analytical specificity, a provisional reference interval, and an explorative method comparison. Results: = 0.88). Conclusion: The test passed the validation, and we now envision the use of the test for exploration of the clinical relevance of specific antithrombin proteoforms. Puzzling cases of antithrombin deficiency, for instance, due to ambiguous activity results or an atypical clinical presentation, can be investigated by the LC-MRM mass spectrometry test serving as an add-on to the activity test and providing a molecular diagnosis. Clinical studies are planned to investigate the potential of the test to improve antithrombin diagnostics. Furthermore, the molecular information gained using the test may aid in establishing better risk stratification and a basis for personalized medicine.

Authors 4

1. Mirjam Kruijt corresponding Aachen Department of Clinical Chemistry Laboratory Medicine

   Leiden University Medical Center

   Affiliation as printed

   Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, the Netherlands

   Leiden University Medical Center, Department of Clinical Chemistry and Laboratory Medicine, Albinusdreef 2, 2333 ZA, Leiden, the Netherlands

2. Maxim M. Treep Aachen Department of Clinical Chemistry Laboratory Medicine

   Leiden University Medical Center

   Affiliation as printed

   Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, the Netherlands

   Leiden University Medical Center, Department of Clinical Chemistry and Laboratory Medicine, Albinusdreef 2, 2333 ZA, Leiden, the Netherlands

3. Christa M. Cobbaert Aachen Department of Clinical Chemistry Laboratory Medicine

   Leiden University Medical Center

   Affiliation as printed

   Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, the Netherlands

   Leiden University Medical Center, Department of Clinical Chemistry and Laboratory Medicine, Albinusdreef 2, 2333 ZA, Leiden, the Netherlands

4. L. Renee Ruhaak Aachen Department of Clinical Chemistry Laboratory Medicine

   Leiden University Medical Center

   Affiliation as printed

   Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, the Netherlands

   Leiden University Medical Center, Department of Clinical Chemistry and Laboratory Medicine, Albinusdreef 2, 2333 ZA, Leiden, the Netherlands