Within this project, we aimed to develop a clinical chemistry test that characterizes AT at the molecular level, analytically validate the method and assess the level of agreement between activity tests, antigen based tests and our molecular test. Mass spectrometry (MS) is a technology that allows for the quantitation of proteins in a multiplexed manner at the molecular level. We used a so-called bottom up proteomics approach, in which the protein of interest is enzymatically digested into representing peptides. These peptides are then quantified based on their specific mass relative to an internal standard. However, if molecular variation in specific aminoacids exists, this is also reflected in their mass, and thus observable by the MS technology. We were able to develop a method that allows for 1. quantitation of AT in molar units, 2. identification of most of the important mutations, 3. distinction of the alpha and beta form of AT based on the glycosylation, and 4. generally altered AT glycosylation. The method was then thoroughly analytically validated according to clinical chemistry guidelines for its precision, linear range, LoD and LoQ, stability and specificity. To assess the potential for harmonization of AT activity tests using our method, AT standards were characterized using intact protein mass spectrometry. However, the standards proved not to be suitable for our intended purpose. We are currently collaborating with the EQA organizer ECAT to develop alternative standards and samples to assess the behaviour of specific well-characterized AT materials in a number of CE-IVD marked AT tests.
Based on this work we were able to present to researchers and clinicians at several (online) conferences, including the annual symposium of the American Society for Mass Spectrmetry, The Dutch Society for Clinical Chemistry and in July 2021 the International Society for Thrombosis and Hemostasis. Moreover, based on our work, we have initiated collaborations with the University of Murcia on the characterization of AT in clinical samples, and within the LUMC, the department of internal medicine, with whom we collaborate on the application of the development strategy established in our work on other proteins in the coagulation cascade. The work has thus far resulted in 2 peer reviewed publications, and we were also able to guide 3 bachelor and master students during their final internships. Due to the COVID pandemic, and the not yet clinical applicability of the current results, the outreach of to the general public has been limited.
