Abstract Number: OC 04.3
Meeting: ISTH 2021 Congress
Background: Antithrombin (AT) testing is typically performed using activity assays. Yet, the molecular variation, resulting in different proteoforms, that may underlie AT deficiency is often neglected. We developed a mass spectrometry method that allows simultaneous AT quantification and characterization of AT proteoforms at the molecular level. A female patient presented with recurrent pregnancy loss and borderline low AT activity levels (69-72%). The responsible physician doubted whether the relatively mildly lowered AT levels were of clinical significance and were based on hereditary deficiency and requested further AT analysis.
Aims: Assess the clinical relevance of a mildly lowered AT activity level in a female patient with recurrent pregnancy loss.
Methods: AT is immunocaptured from (50 μL 100x diluted) citrated plasma and is subsequently denatured, alkylated and tryptically digested. Proteotypic peptides covering 49,6% of the total AT protein are quantified by mass spectrometry. The total assay can be executed in less than six hours and is semi-automated. Using an external calibration curve, AT mass is quantified in the samples, and in the same test AT is molecularly characterized through monitoring of specific peptides. If alterations are observed, additional testing can further identify specific mutations.
Results: Our lab-developed-test identified unequivocally a type-IIb AT deficiency in this patient. Through further examination of the affected peptide, the exact mutation was identified as a Pro73Leu mutation (mutation Basel), which is known to provide unequivocal results in activity assays. The mutation is also associated with obstetric complications.
Conclusions: This case study shows the potential of mass spectrometry-based protein testing for quantifying the antigen concentration of AT and simultaneously identifying a type-IIb mutation using a single test. It highlights the ability of bottom up proteomics testing to contribute to precision diagnostics, for the sake of improved patient care.
To cite this abstract in AMA style:Kruijt M, van der Pol LM, Eikenboom J, Verburg HJ, Cobbaert CM, Ruhaak LR. Unraveling Mild Antithrombin Deficiency with Mass Spectrometry in a Patient Presenting with Recurrent Pregnancy Loss [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/unraveling-mild-antithrombin-deficiency-with-mass-spectrometry-in-a-patient-presenting-with-recurrent-pregnancy-loss/. Accessed March 4, 2024.
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