Academy of Diagnostics & Laboratory Medicine - Scientific Short
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M-protein isotyping has traditionally been performed using immunofixation electrophoresis (IFE) in clinical laboratories. With IFE, proteins are separated by electrophoresis according to overall charge, followed by immunoprecipitation with immunoglobulin heavy- and light-chain–specific antisera. Although widely used, IFE is manual, labor-intensive, time-consuming, and heavily dependent on expert interpretation.
The MASS-FIX assay, implemented clinically in 2018, couples immunoaffinity enrichment with MALDI-TOF mass spectrometry to detect and characterize M-proteins in serum and urine1. Unlike IFE, which relies on electrophoretic separation, MASS-FIX identifies M-proteins by their unique light-chain mass-to-charge (m/z) signatures. MASS-FIX is found to be more sensitive and provides quantitative information on M-protein levels. The limit of quantitation (LOQ) of MASS-FIX is set at 10 mg/dL, while the limit of detection (LOD) can be as low as 1 mg/dL. MASS-FIX identifies M-proteins by their masses, a feature that makes it particularly useful for disease monitoring by focusing on a specific clone or detecting subtle shifts in clonal populations. Currently, a clone with a mass within ± 20 Daltons of the original clone is considered the same clone in the follow-up testing. Furthermore, MASS-FIX can be automated and is less reliant on subjective interpretation, thereby reducing inter-observer variability.
The use of m/z signatures has also uncovered features of M-proteins not previously well appreciated. Approximately 5% of M-proteins demonstrate light-chain glycosylation, which increases the light-chain mass by up to 1,200 Da. Importantly, light-chain glycosylation has been shown to be an independent risk factor for progression to symptomatic disease. Specifically, glycosylated light chains associated with IgG or IgA are linked to a higher risk of progression to amyloid light-chain (AL) amyloidosis, whereas glycosylated light chains associated with IgM indicate an increased risk of progression to cold agglutinin disease2.
More recently, studies have focused on the detection of light-chain fragments by MASS-FIX. These fragments, which appear at markedly lower masses than intact light chains, are observed in up to 1% of M-protein–positive patients. Our investigations of patients with light-chain fragments demonstrate that these fragments are produced in vivo and remain attached to the M-protein heavy chain, hence why they are not observed by traditional electrophoretic methods. Interestingly, light-chain fragmentation is more common when the M-protein is of a lambda isotype. Given that AL amyloidosis is also lambda skewing, examination of clinical association demonstrated that patients with detectable light-chain fragments had an increased odds ratio of 2.68 for AL amyloidosis. Further population-based studies are needed to confirm this association.
As MASS-FIX and related technologies move toward replacing traditional methods, a central challenge for clinicians and laboratorians will be how to translate these novel findings into clinical practice to improve patient care. With clinical association studies of the above-mentioned novel M-protein features detected by MASS-FIX, more specific recommendations for follow-up testing or monitoring plans may be incorporated into the laboratory reports accompanying the test results.
