Mauch, Emily

Monoclonal gammopathy of thrombotic significance (MGTS) is a recently recognized thrombophilia characterized by the production of a monoclonal antibody that binds to platelet factor 4 (PF4) and stimulates platelet activation (Blood. 2022 Jul 7;140(1):73-77). Typical signs of MGTS include a history of recurrent thrombosis, persistent thrombocytopenia, and positive laboratory results in PF4/polyanion enzyme-linked immunosorbent assay (ELISA) and serotonin release assay (SRA) testing, as in the related thrombotic disorder heparin-induced thrombocytopenia (HIT). Here, we present a case of MGTS, presenting with transient thrombocytopenia coincident with thrombotic episodes and negative test results in SRA and PF4/polyanion-based testing platforms (ELISAs and automated assays). A 56-year-old male patient initially presented with acute left lower extremity deep vein thrombosis (DVT) and a pulmonary embolism. Following the administration of warfarin, the patient experienced a second DVT four months later and was transitioned to a factor Xa inhibitor. Fifty-two months later, the patient developed occlusion of the splenic and superior mesenteric veins despite additional anticoagulation therapy. Further testing led to a diagnosis of an IgG2 kappa monoclonal gammopathy of undetermined significance (MGUS). Eleven months later, the patient again suffered breakthrough thrombosis, specifically radial artery thrombosis, and a thrombotic cerebrovascular accident, leading to the addition of aspirin to the patient's treatment regimen. Notably, where detailed blood counts were available in the patient's clinical history, the patient experienced only transient thrombocytopenia coinciding with his two most recent thrombotic episodes and has had platelet counts in the normal range in the four-year period since his last thrombotic event. After initiating dual anticoagulant/antiplatelet therapy, the patient has not developed recurrent thrombocytopenia or new thrombotic events. Antigen-based and functional testing for HIT antibodies (ELISA, SRA, and automated immunoassays) were negative in this patient, unlike in the previously described index case of MGTS, where serological testing mimicked classical HIT serology (i.e., HIT ELISA and SRA positivity). The sample was also non-reactive in a vaccine-induced immune thrombotic thrombocytopenia (VITT)-specific ELISA employing uncomplexed PF4 targets, as previously described (Am J Hematol. 2022 May;97(5):519-526). Conversely, the patient's antibody consistently stimulated platelet activation in a PF4- and IgG-dependent manner in functional testing using PF4-treated platelets. This included PF4-treated platelets that were freshly isolated and used in the PF4-dependent P-selectin expression assay (PEA) and cryopreserved platelets used in the thrombospondin-1 release assay. Interestingly, consistently positive PEA results have been documented from four samples over the last four years, during which the patient had normal platelet counts. To further characterize the patient's IgG2 MGUS antibody, the patient's monoclonal was immuno-enriched. Patient sera and the immuno-enriched MGUS were then compared by mass spectrometry, which generated identical mass profiles. After confirming the effective enrichment of the patient's MGUS by mass spectrometry, the antibody was de novo sequenced or “reverse-engineered” to obtain the antibody's amino acid sequence. The antibody was then expressed and tested in ELISA-based and functional platelet assays. Importantly, the recombinant antibody replicated the PF4-dependent platelet activation observed with patient serum and did not bind PF4/polyanion targets in ELISA testing. Together, these data strongly support the identification of a new class of ELISA- and SRA-negative anti-PF4 monoclonal antibodies that cause MGTS. This case illustrates the diagnostic challenges in MGTS. MGTS patients may not exhibit persistent thrombocytopenia, and diagnostic tests for detecting HIT antibodies may be ineffective in accurately diagnosing this new thrombophilia. This case emphasizes the importance of reviewing platelet counts in the peri-thrombotic period to recognize transient thrombocytopenia and establishes the utility of PF4-enhanced functional assays to detect pathogenic anti-PF4 antibodies, even in settings where classical HIT testing (i.e., ELISA/SRA) is negative.

We identified a patient with a recently described syndrome, monoclonal gammopathy of thrombotic significance (MGTS; Blood. 2023 Apr 6;141(14):1772-1776), and three additional patients with high 4Ts scores who were consistently negative in the PF4-polyanion HIT ELISA (Lifecodes PF4 IgG, Immucor). The MGTS patient, with a monoclonal IgG kappa, had recurrent thrombosis, and transient mild thrombocytopenia (nadir in the low 100s) over a period of years, and was noted to have a HIT ELISA optical density (OD) of 0.117. The three HIT patients experienced thrombocytopenia and thrombosis with ELISA ODs of 0.37, 0.18 and 0.20. All patients were positive in platelet-activation (functional) testing demonstrating PF4 dependence and inhibited with both high concentrations of heparin (100U/mL) and the FcγRIIa blocking monoclonal antibody (mAb) IV.3, consistent with the presence of anti-PF4 antibodies in the patient samples ( data not shown). Because the serologic profile of these patients suggested a novel HIT subtype characterized by negative HIT ELISA results, we undertook a functional platelet assay screen of 500 consecutive HIT ELISA-negative patients tested in our diagnostic laboratory. Research studies were approved by the Institutional Review Board of Mayo Clinic. Of the 500 patients tested, 20 (4.0%) stimulated platelet activation in the PF4-dependent P-selectin Expression Assay (PEA ≥19%; data not shown). To exclude non-specific activation that can be induced by HLA antibodies, additional testing was performed to evaluate whether platelet activation was PF4-dependent using buffer or PF4-treated platelets. Three patients demonstrated PF4-dependent activation that was inhibited by high heparin concentrations and mAb IV.3, as would be expected for HIT antibodies (ELISA-negative HIT, EN-HIT1-3; Figure 1). On retrospective review, all three patients thus identified had clinical histories and courses consistent with high clinical probability for HIT. To develop tools to study this new class of antibodies, monoclonal HIT antibodies were generated in mice after PF4/heparin immunizations and screened using a novel approach that included simultaneous testing of hybridoma supernatants in the PEA and PF4-polyanion ELISA. This strategy was designed to enabled us to identify mAbs that activated platelets (in the PEA), but that did not bind antigenic targets in the HIT ELISA. A novel mAb 431.4 that was developed using this approach, and the well-characterized HIT-like mAb KKO were evaluated for their abilities to both bind PF4-polyanion targets in ELISA, and to activate PF4-treated platelets in the PEA (Figure 2). In significant contrast to KKO, mAb 431.4 did not bind PF4/polyanion complexes, however, like KKO, strongly activated platelets in a PF4-dependent manner. Activation induced by both KKO and mAb 431.4 was inhibited by high heparin concentrations, as would be expected for HIT antibodies. In vivo murine studies using this novel mAb are pending at this time. In summary, the non-canonical diagnostic profile of HIT ELISA-negative but platelet activation-positive antibodies were found at a relatively high incidence in HIT-suspected patients (0.6%; 3 of 500 ELISA-negative patients). These findings have important diagnostic/management implications and suggest a re-evaluation of ASH's HIT management guidelines as noted in recommendations 2.7 and 2.8 of that document (“In patients with an intermediate (Recommendation 2.7) or high (Recommendation 2.8)-probability 4Ts score and a negative immunoassay, the ASH guideline panel recommends discontinuation of the non-heparin anticoagulant and resumption of heparin, if indicated ”; Blood Adv (2018) 2 (22): 3360-3392). Figure 1. A subset of ELISA-negative HIT-suspected patients harbor anti-PF4 platelet-activating antibodies. Three ELISA-negative (EN) HIT patient samples from a screen of 500 samples demonstrate PF4-dependent platelet activation and inhibition with high heparin concentration and mAb IV.3 incubation. PBS, phosphate buffered saline; IC, isotype control; HDH- high dose heparin (100U/mL). Figure 2. Murine mAb 431.4 serologically mimics pathogenic ELISA-negative HIT/MGTS antibodies. ELISA and PEA testing was performed using the “HIT-like” mAb KKO, novel mAb 431.4, or an isotype control (IC) antibody. PVS-polyvinyl sulfonate (polyanion used in the Immucor IgG ELISA assay).