What does mass spectrometry add to serum free light chain testing?

According to the International Myeloma Working Group, the serum free light chain (FLC) assay is an important prognostic marker for monoclonal gammopathies, including monoclonal gammopathy of uncertain significance (MGUS), smoldering myeloma, multiple myeloma, solitary plasmacytoma, and AL amyloidosis [1, 2]. The measurement of FLCs is especially valuable for identifying and monitoring treated patients with AL amyloidosis and those with non-secretory or oligosecretory myeloma, where traditional electrophoresis-based methods have fallen short due to limited sensitivity. The most widely used methods of determining total FLCs are immunoassays, which use a ratio between the total kappa and total lambda FLCs to infer clonality. Like other immunoassays, FLC immunoassays can suffer from the hook effect, lot-to-lot variation in the reagent antisera [3], and artifacts arising from FLC aggregation [2]. However, the core limitation of FLC immunoassays is that they do not directly measure clonality. This means that the interpretation of clonality is heavily reliant on the reference interval, which is not easily established or generalizable and has been the topic of much discussion [3, 4]. Specifically, in patients with elevated polyclonal antibody backgrounds, such as those with chronic kidney disease, infection, or autoimmune disease, the kappa to lambda FLC ratio can be skewed and hard to interpret [5-7].

In the past few years, a wave of studies has demonstrated the utility of mass spectrometry (MS) in addressing this issue. This field was pioneered by a group of scientists at the Mayo Clinic, who developed a MALDI-TOF-based method for measuring serum monoclonal proteins (MASS-FIX) [8]. In 2019, Sepiashvili et al. demonstrated that MASS-FIX can be applied for FLCs, showing that it can directly determine the clonality of FLCs within elevated polyclonal antibody backgrounds with markedly improved analytical sensitivity compared to immunofixation electrophoresis [9]. On the other hand, around 16% of samples with negative electrophoresis and MASS-FIX results had abnormal results using FLC immunoassay [3, 4]. More recently, Yeung et al. published a method that combines an on-probe immunocapture step coupled with high-resolution MS (OPEX-MS) to determine the clonality of unmodified FLCs [10]. They identified monoclonal FLCs in 14% of immunoassay-negative samples but also found that 33% of kappa elevated samples as well as 83% dual kappa and lambda FLC elevated samples by immunoassay did not have any monoclonal FLCs by OPEX-MS [10]. Both studies reported a ~10% prevalence of glycosylated light chains in their tested cohorts [9, 10].

Together, the findings reported by using MS-based methods show that:

1. The direct detection of all FLCs allows for clonality determination without relying on a reference interval for the ratio of total kappa and lambda FLCs.
2. The improved analytical sensitivity compared to immunoassay makes these methods valuable in monitoring patients who have been treated to a low level of residual disease or those with oligosecretory disease.
3. There are often no monoclonal proteins in samples with mildly elevated or dual elevated FLC immunoassay results, which makes MS a useful tool in confirming clonality prior to rendering a diagnosis of light chain MGUS in these patients.
4. Because light chain glycosylation status has been demonstrated to have prognostic value [11], it can be used in combination with other established factors for patient risk stratification. Information about post-translational modifications is not accounted for in currently available immunoassays and may even hinder their assay performance.

These advantages highlight the value of MS in serving as a complementary testing method to FLC immunoassays in evaluating patients with monoclonal gammopathies. Whereas immunoassays can quantify kappa and lambda FLCs, MS is helpful in determining clonality and identifying informative post-translational modifications.

References

1. Rajkumar, S.V., Multiple myeloma: 2022 update on diagnosis, risk stratification, and management. Am J Hematol, 2022. 97(8): p. 1086-1107.
2. Dispenzieri, A., et al., International Myeloma Working Group guidelines for serum-free light chain analysis in multiple myeloma and related disorders. Leukemia, 2009. 23(2): p. 215-24.
3. Rozenova, K., et al., Kappa Free Light Chain Drift Prompts the Need for a New Upper Limit of Normal Free Light Chain Ratio to Avoid an Epidemic of Kappa Light Chain Monoclonal Gammopathy of Undermined Significance. J Appl Lab Med, 2023. 8(4): p. 742-750.
4. Willrich, M.A.V., Mass Spectrometry Meets Free Light Chains: A Path toward Greater Diagnostic Precision. Clin Chem, 2024.
5. Long, T.E., et al., Defining new reference intervals for serum free light chains in individuals with chronic kidney disease: Results of the iStopMM study. Blood Cancer J, 2022. 12(9): p. 133.
6. Lutteri, L. and J.F.M. Jacobs, Reference ranges of the Sebia free light chain ratio in patients with chronic kidney disease. Clin Chem Lab Med, 2018. 56(9): p. e232-e234.
7. Sprangers, B., et al., Comparison of 2 Serum-Free Light-Chain Assays in CKD Patients. Kidney Int Rep, 2020. 5(5): p. 627-631.
8. Barnidge, D.R., et al., Monitoring free light chains in serum using mass spectrometry. Clin Chem Lab Med, 2016. 54(6): p. 1073-83.
9. Sepiashvili, L., et al., Direct Detection of Monoclonal Free Light Chains in Serum by Use of Immunoenrichment-Coupled MALDI-TOF Mass Spectrometry. Clin Chem, 2019. 65(8): p. 1015-1022.
10. Yeung, P.S.W., et al., Clonality Determination by Detecting Unmodified Monoclonal Serum Free Light Chains Using On-Probe Extraction Coupled with Liquid Chromatography-High-Resolution Mass Spectrometry. Clin Chem, 2024.
11. Dispenzieri, A., et al., N-glycosylation of monoclonal light chains on routine MASS-FIX testing is a risk factor for MGUS progression. Leukemia, 2020. 34(10): p. 2749-2753.