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Monoclonal gammopathy of thrombotic significance is a prothrombotic condition characterized by platelet-activating monoclonal anti-platelet factor 4 (PF4) antibodies1. Hallmark findings include a history of recurrent thrombosis, persistent thrombocytopenia, and positivity in classical heparin-induced thrombocytopenia (HIT) assays such as the PF4/polyanion enzyme-linked immunosorbent assay (ELISA) and serotonin release assay (SRA)1,2. Here, we present the diagnostic challenges related to an ELISA/SRA-negative monoclonal gammopathy of thrombotic significance patient who presented with recurrent breakthrough thrombosis accompanied by only transient thrombocytopenia coincident with thrombotic episodes. A 56-year-old male developed unprovoked acute left lower extremity deep vein thrombosis (DVT) and pulmonary embolism (Time 0, Fig 1A). He was started on warfarin but developed another DVT several months later (Fig 1A) and was transitioned to rivaroxaban. Despite anticoagulation therapy, the patient developed occlusion of the superior mesenteric and splenic veins (Fig 1A) and shortly thereafter was found to have a monoclonal gammopathy of undetermined significance (MGUS) comprising an IgG2 kappa antibody (1.8 gm/dL). Subsequently, the patient also experienced radial artery thrombosis and a cerebrovascular accident (CVA), at which time aspirin was added to his treatment regimen (Fig 1A & B). Platelet counts, available during his two most recent thrombotic episodes, demonstrated only a transient decrease coinciding with thrombotic events (Fig 1B).

the patient also experienced radial artery thrombosis and a cerebrovascular accident (CVA), at which time aspirin was added to his treatment regimen (Fig 1A & B). Platelet counts, available during his two most recent thrombotic episodes, demonstrated only a transient decrease coinciding with thrombotic events (Fig 1B). A spontaneous HIT diagnosis was entertained but was not supported by either ELISA or SRA testing (Fig 1B & C), including testing in an automated, latex bead-based HIT assay (HemosIL HIT-Ab [PF4-H]) and an uncomplexed PF4 ELISA specific for VITT antibodies3 (Fig S1). However, multiple patient samples obtained over two years demonstrated consistent PF4-dependent platelet activation in the PF4-dependent p-selectin expression assay (PEA; Fig 1B) and also activated PF4-treated cryopreserved platelets in a recently developed thrombospondin-1 release assay4 (Fig S2). During this period of persistent PEA positivity, no thrombocytopenia was seen (Fig 1B). Platelet activation induced by patient samples were inhibited by high concentrations of heparin and blockade of the FcGRIIa receptor, as would be expected for anti-PF4 antibodies (Fig 1D). Immunoglobulin G (IgG) antibodies from the patient’s serum also bound platelets in a PF4-dependent manner, similar to HIT sera (Fig 1E). This antibody was neither HIT nor VITT-like (Table S1). The MGUS antibody was isolated, validated by mass spectrometry5 (Fig S3), de novo sequenced, and produced recombinantly. The recombinant antibody bound to PF4/polyanion complexes to a greater extent than pooled human IgG (Intravenous immunoglobulin G, IVIg; Fig 1F) and demonstrated both PF4-dependent platelet activation (Fig 1G) and platelet binding (Fig 1H), recapitulating testing results with patient serum.

and produced recombinantly. The recombinant antibody bound to PF4/polyanion complexes to a greater extent than pooled human IgG (Intravenous immunoglobulin G, IVIg; Fig 1F) and demonstrated both PF4-dependent platelet activation (Fig 1G) and platelet binding (Fig 1H), recapitulating testing results with patient serum. Together, these data suggest that ELISA/SRA-negative monoclonal gammopathy of thrombotic significance, an entity that can cause severe recurrent breakthrough thrombosis, can be challenging to diagnose. The disorder may occur without persistent thrombocytopenia and requires PF4-enhanced platelet-based testing for confirmation.