MSACL 2025 Abstract

A Molecular Approach to Antithrombin Deficiency: Next-Generation Diagnostics by Mass Spectrometry

N. Kijk in de Vegte (1), M. van der Helm (1), M.E. de la Morena-Barrio (2), Z. Bereczky (3), J. Corral (2), C.M. Cobbaert (1), L.R. Ruhaak (1)
(1) Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, The Netherlands (2) Servicio de Hematología, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII, Murcia, Spain (3) Division of Clinical Laboratory Science, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary

Niek Kijk In de Vegte, MSc (Presenter) Leiden University Medical Center

Presenter Bio: I am a second-year PhD candidate at the Leiden University Medical Center in the Netherlands, specializing in precision diagnostics within the Department of Clinical Chemistry and Laboratory Medicine. My research focuses on developing and validating LC-MRM-MS-based assays for the molecular characterization of antithrombin (AT) deficiencies, with the goal of improving diagnostic accuracy and patient care.

By integrating mass spectrometry with a bottom-up proteomics approach, my work enables both quantitative and structural analysis of AT proteoforms. This novel diagnostic method allows for the identification of AT variants, differentiation between ATD subtypes, and detection of glycosylation abnormalities that remain undetected in conventional activity-based assays.

I work closely with clinical experts to translate these molecular insights into practice, particularly in the context of venous thrombosis risk assessment and recurrent miscarriages. Through this interdisciplinary collaboration, I aim to bridge the gap between laboratory innovations and personalized medicine, ultimately contributing to improved patient outcomes in thrombosis care.

Relevant Financial Disclosures (within past 24 months, reported on May 26, 2025) No relevant financial relationship(s) to disclose.

INTRODUCTION:
Current antithrombin deficiency (ATD) diagnostics rely on functional activity assays, which provide limited molecular insight and may lead to misclassification of patients. In line with the principles of P5 medicine (preventive, personalized, predictive, participatory, and psychocognitive), a more precise approach is needed to improve diagnosis and patient management. Given the clinical heterogeneity of ATD, we developed a mass spectrometry (MS)-based test that enables both quantitative and molecular characterization of antithrombin proteoforms in patient plasma. This test aims to identify antithrombin (AT) variants, differentiate between ATD subtypes, and provide a deeper understanding of AT (patho-) biology. Here, we evaluate the analytical and clinical performance of this next-generation MS-based diagnostic approach.

METHODS:
An LC-MRM-MS-based test enabling both accurate quantitation and molecular characterization of AT through a bottom-up proteomics approach was developed and analytically validated according to ISO 15189 for medical laboratories. Immunocapture was performed using a biotinylated anti-AT antibody coupled to a streptavidin-coated plate, allowing specific enrichment of AT from plasma. Following capture, AT was reduced with TCEP, alkylated with iodoacetamide, and digested with trypsin. A stable isotope-labeled (SIL) peptide mix was included as an internal standard for accurate quantitation. The assay measured 23 AT-derived peptides, monitoring a total of 126 transitions in dynamic multiple reaction monitoring (dMRM) mode, with one quantifying transition and one or two qualifying transitions per peptide. Data were acquired on an Agilent 6495 triple quadrupole mass spectrometer coupled to an Agilent 1290 UPLC system. A semi-automated data analysis workflow was developed using a dedicated R-script, providing a binary output for clinical interpretation. System suitability was ensured using a peptide-based test sample and QC monitoring to validate instrument performance and assay precision.

Plasma samples from two independent patient cohorts were analyzed: (1) 91 patients with functionally and genetically characterized ATD and 41 patients with congenital disorders of glycosylation affecting AT glycosylation; (2) 71 individuals carrying the AT Budapest3 mutation and 46 healthy controls. Scientific validity of the test was evaluated in relation to the genetic background.

RESULTS:
The MS test quantifies 23 AT peptides, achieving a mean within-laboratory imprecision of 5.9% and a linear range of 0.08–2.58 µmol/L. The reference interval was established at 1.07–1.49 µmol/L. A dedicated R-script was developed for automated data analysis, providing a binary output suitable for clinical interpretation.

In the first cohort of 91 patients with ATD due to SERPINC1 defects, the MS test demonstrated a diagnostic sensitivity of 100%, compared to 81.1% for functional activity assays overall and 56.8% for type II ATD. The test successfully identified type I and type II ATD, including heparin-binding site (HBS) mutations such as p.Pro73Leu (AT Basel), p.Leu131Phe (AT Budapest3), and p.Arg79His (AT Padua), which are often missed by activity-based assays. Additionally, in patients with borderline activity levels, the MS test correctly classified cases that would otherwise remain in a diagnostic “grey zone,” reducing the likelihood of misclassification. In a subgroup of 41 patients with congenital disorders of glycosylation, MS analysis revealed a significantly lower proportion of the α-AT proteoform (71.1% vs. 94.6% in controls). Further glycopeptide analysis identified altered glycosylation at N224, highlighting the test’s ability to detect glycosylation abnormalities that remain undetected in activity assays.

In the second cohort, the MS test successfully distinguished homozygous and heterozygous AT Budapest3 (ATBp3) carriers from healthy controls by detecting quantitative deficiencies and altered proteoform distribution. The GP-LGACN ratio, a key biomarker for ATBp3, was significantly lower in all ATBp3 patients, with values below a threshold of 0.82, enabling clear molecular differentiation.

To confirm mutations in patients with AT deficiency, variant specific transitions were established for >20 mutations, including p.Val30Glu (AT Dublin), p.Arg45Trp, p.Pro73Leu (AT Basel), p.Leu131Phe (AT Budapest3), p.Arg177Cys, p.Arg425Cys.

CONCLUSION:
The developed next generation MS-based diagnostic AT test offers both qualitative and quantitative insights into AT proteoforms, refining the diagnostic landscape for ATD. Through molecular-level characterization, current limitations of activity assays are overcome, enabling personalized risk stratification and precision diagnostics in line with P5 medicine principles. The detection of clinically relevant AT variants and glycosylation abnormalities through the MS-based test is envisioned for use as an add-on tool in cases of diagnostic uncertainty. Future studies integrating clinical outcomes and our molecular data are necessary to optimize predictive and preventive strategies for ATD management. If successful, our precision lab-developed test promises to refine AT-diagnosis, alter the clinical pathway for antithrombin deficiency and consequently improve patient management.