Unraveling a borderline antithrombin deficiency case with quantitative mass spectrometry

Abstract

Antithrombin deficiency diagnostics by first-line activity tests suffer from a lack of sensitivity sometimes resulting in diagnostic uncertainty. We here present a case of a woman with recurrent pregnancy loss who was screened for inherited thrombophilia. Antithrombin activity was borderline low, resulting in uncertainty about the correct diagnosis. Using a mass spectrometry-based test, the antithrombin protein of the patient was characterized at the molecular level and a heterozygous p.Pro73Leu mutation was identified. The mutation, also known as antithrombin "Basel," increases the risk of venous thromboembolism and obstetric complications. This case is illustrative of current antithrombin deficiency screening, in which diagnoses may be missed by traditional diagnostics. Next-generation protein diagnostics by mass spectrometry provides molecular insight into the proteoforms present in vivo. This information is essential for laboratory specialists and clinicians to unambiguously diagnose patients and will aid in evolving healthcare from traditional to precision diagnostics.

Keywords: antithrombin; antithrombin deficiency; mass spectrometry; molecular characterization; next-generation protein diagnostics; recurrent pregnancy loss.

FIGURE 1

Current and proposed recommendation for antithrombin (AT) deficiency screening. A, Current strategy for patients suspected to have a thrombophilia. An AT activity test is performed but this test may generate ambiguous results. It is up to the clinician to decide whether results have clinical significance potentially leading to missed diagnoses. B, Results for AT activity tests (upper table) and liquid chromatography coupled to multiple‐reaction‐monitoring mass spectrometry (LC‐MRM‐MS) test (lower table and graphs). Colored results in the tables indicate abnormalities. Low concentration for peptide IPEATNR in combination with the presence of mutated peptide ILEATNR ascertains a heterozygous p.Pro73Leu mutation. C, Proposed strategy for patients suspected to have a thrombophilia with ambiguous AT activity results. Using next‐generation protein diagnostics, we provide a molecularly defined diagnosis to aid both clinician and patient in the diagnostic process for AT deficiency