Kanack, Adam

ABSTRACT Background Monoclonal gammopathy of thrombotic significance (MGTS) is a recently described chronic prothrombotic condition characterized by monoclonal anti-PF4 antibodies that are detected above the polyclonal antibody background in patient sera (i.e. present as monoclonal gammopathy of undetermined significance, MGUS). Due to conflicting data in the published literature on antibody clonality in heparin-induced thrombocytopenia (HIT), we evaluated clonality and abundance of anti-PF4 antibodies in HIT, including investigating whether an MGUS, if present in HIT, represents the causative anti-PF4 antibody. Methods Blood samples from 15 patients with HIT were subject to Platelet Factor 4-dependent antigen-based and functional tests. The unmanipulated serum antibody repertoire and isolated anti-PF4 antibodies were subjected to mass spectrometric evaluation. Results Two of the 15 HIT patients had an IgG MGUS. Notably, anti-PF4 antibodies were not synonymous with the MGUS antibody in either of the two patients. Eight of the 15 patients demonstrated monoclonal anti-PF4 antibodies, however, none of the anti-PF4 antibodies were detectable as an MGUS upon evaluation of the entire serum antibody repertoire, reflecting their low abundance. In the seven patients with multiple anti-PF4 antibodies, non-monoclonality was confirmed by analysis of deglycosylated antibody heavy chains. Conclusions Anti-PF4 HIT antibodies are monoclonal in approximately 50% of HIT patients, however, antibody abundance is low such that they are not detectable over the polyclonal IgG background (i.e. are MGUS-negative), differentiating HIT from MGTS. This observation helps explain the transient nature of HIT relative to the persistent prothrombotic state seen in MGTS.

Abstract Vaccine‐induced immune thrombotic thrombocytopenia (VITT) is a new disorder that emerged in the wake of coronavirus disease 2019 (COVID‐19) vaccination. It is a rare but life‐threatening condition that requires an aggressive course of treatment to improve patient outcomes. Currently, the diagnosis of VITT relies on a comprehensive panel of criteria, including a history of recent vaccination, platelet count, positive enzyme linked immunosorbent assays (ELISA) result for a closely related thrombotic disorder, heparin‐induced thrombocytopenia (HIT), and platelet factor 4 (PF4)‐dependent functional assays. The study describes a technically simple antigenic assay designed to detect autoimmune antibodies associated with VITT. It is first shown that cross‐linked platelet factor 4 (c‐PF4) represents an antigenic target specific for VITT/VITT‐like Abs. This antigenic target is then incorporated into a microfluidic electrochemical biosensor and specific and sensitive detection of VITT/VITT‐like antibodies is demonstrated in a fully automated manner while using microliter volumes of patient sera. Fifty‐five serum samples are tested using the microfluidic electrochemical biosensor and demonstrated high accuracy for the detection of VITT sera and VITT‐like monoclonal gammopathy of thrombotic ignificance (MGTS) antibodies compared to healthy controls and HIT patients. It is envisioned that the microfluidic electrochemical biosensor will be used in combination with other clinical criteria to enable timely, sensitive, and specific diagnosis of VITT.