Technology Spotlight
Use of Mass Spectrometry to Study Antibody Clonality Leads to Identification of Novel Thrombophilia: Monoclonal Gammopathy of Thrombotic Significance

Monoclonal gammopathy of clinical significance (MGCS) is a general descriptor of symptomatic diseases that are related to a monoclonal antibody (M-protein).¹  These diseases differ from traditional, asymptomatic monoclonal gammopathy of undetermined significance (MGUS), as the M-protein is suspected or implicated in disease pathogenesis. Associating the M-protein with causation of signs and symptoms of a presenting disorder is not a straightforward endeavor. MGUS is a relatively common finding, as 3% to 4% of people over the age of 50 will have an M-protein.²  MGUS also represents an immune dysfunction, and when a person has a coexisting autoimmune disease, it is difficult to separate the immune dysfunction from the M-protein as the cause of the symptoms.

Mass spectrometry (MS) offers a means to better evaluate the M-protein as the cause of disease. MS-based detection, isotyping, and quantitation of M-proteins has become available in a few clinical laboratories.³  These methods rely on the mass accuracy and increased resolution of MS instrumentationto provide increased sensitivity and specificity of M-proteins in myeloma patients.⁴  These same features are also being used to examine antibodies to determine isotype and clonality (i.e., monoclonal, oligoclonal,or polyclonal) in patients with autoantibodies. As a prime example, recent MS characterization of anti-platelet factor 4 (PF4) antibodies have shed new light on the clonality and isotype of these autoantibodiesin patients with heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombotic thrombocytopenia.⁵  These studies have also demonstrated that PF4-specific M-proteins can have platelet-activating properties causing thrombosis — an entity we have termed monoclonal gammopathy of thrombotic significance (MGTS).

To establish the presence of an M-protein, apatient’s serum is separately immuno-enriched for immunoglobulins (Igs) G, A, M, kappa, and lambda. The enriched Igs are then chemically reduced to produce heavy and light chains, which are subsequently analyzed by MS to produce mass-to-charge (m/z) distribution for each immuno-enrichment. Thelight chain m/z distribution can be examined for over-expressed Igs. Healthy patients’ light chainmass distributions are relatively broad due to the polyclonal Ig background, with no discernable peaks above the background. Peaks above this polyclonal background represent Igs with production rates that are higher than those of other Igs.⁶  By comparing peaks of similar mass in each immuno-enrichment, the number, isotype, mass, and relative abundance can be ascertained.

Figure 1 highlights the basic workflow to evaluate the role of an M-protein in MGTS. First, anti-PF4 antibodies are immuno-enriched from patient’s serum using PF4-coated beads. The bound anti-PF4 antibodies can then be eluted from PF4 beads and immuno-enriched for the expected isotype (IgG, Ig kappa, and Ig lambda). (Of note, platelets express the activating IgG receptor FcγRIIa). MS measurement can provide the number, light chain mass, and isotypes of the anti-PF4 antibody, and the isotype and mass of the anti-PF4 antibody can be compared to the M-protein from serum. In addition to the MS measurements, it is useful to show thatthe isolated anti-PF4 antibody has functional activityin platelet activation studies and that the immune-enriched anti-PF4 antibodies recognize the target antigen.

Recently, the above method was applied to a 64-year-old man with recurrent thrombotic episodes with an IgG kappa M-protein.⁷  MS measurement of the M-protein and anti-PF4 antibodies demonstrated near-identical mass of both the kappa light chain for the M-protein and the anti-PF4 antibodies (Figures 2 and 3; see published paper for IgG heavy chain comparison.⁷ ) The isolated anti-PF4 antibodies were shown to activate platelets and bind PF4 targets. Thus, the patient’s M-protein was implicated as the source of the anti-PF4 activity establishing the diagnosis of MGTS, which can have important treatment implications.

Upon dissemination of the index case,⁷  a case was brought to our attention of a 67-year-old man with recurrent thrombotic disease, who we demonstrated had MS-proven MGTS.⁸  Due to life-threatening episodes of thrombosis despite maximal anticoagulation therapy, the patient was treated with plasma-cell– directed therapy (daratumumab, bortezomib, and dexamethasone), resulting in a prompt response from both clinical and laboratory perspectives (e.g., platelet counts, HIT serology, and M-protein level).⁸  The patient has been in remission for almost a year following treatment intervention.

In summary, MS-based antibody characterization has greatly aided in the implication of the anti-PF4 M-protein in the pathogenesis of MGTS. The general method used to establish this novel thrombophilia will likely be extended to other MGCS entities using other antigen targets, which will, in turn, allow for new treatment modalities to be explored in these patients.