For Immunoglobulin Light Chains, It's Time to Fly!
For decades, the clinical laboratory has exploited the clonal nature of the immune response to detect myeloma and MGUS by using serum protein electrophoresis (SPE). When serum from a healthy patient undergoes electrophoretic separation, the fraction containing the patient's immunoglobulin shows a gaussian distribution, representing the products of thousands of different plasma cell clones, each with a unique electrophoretic mobility. When the serum is from a patient with multiple myeloma, however, every medical student knows that a spike appears instead of the normal diffuse pattern, because one monoclonal protein with only one electrophoretic mobility predominates.
We now have new therapies for myeloma, so it is increasingly important to identify patients with MGUS as early as possible. We usually find them only by chance when astute caregivers request an SPE test in patients with vague signs and symptoms. M-components in MGUS are often easy to see. But how can we catch those really early ones that are still hiding, sometimes in plain sight?
Over the past decade, we have tried several new approaches to protein electrophoresis in the hope that we can better detect small abnormal monoclonal immunoglobulins. High-resolution agarose gels (with enhanced electroendosmosis) and capillary zone electrophoresis (CZE) provide better separation of the serum protein fractions, and this helps a great deal. Immunofixation electrophoresis (IFE), traditionally a confirmatory technique, has been promoted as a first-line screen. It can highlight a low-concentration band in the y region that would otherwise be overlooked, and can also expose M-components hiding behind one of the normal a or p bands, making it perhaps the most sensitive way to screen patients. But it adds time and expense.
Recently, a test that does not rely on electrophoresis at all has challenged the role of SPE or CZE. Sensitive immunoassays specific for free immunoglobulin light chains can help identify unbalanced immunoglobulin production. The serum free light chain ratio (FLCR) test was originally used when disorders caused by significant monoclonal free light chain production (such as amyloidosis) were suspected. But it is now clear that an abnormal serum FLCR may precede the appearance of an abnormal band by SPE or CZE (or even IFE) in many patients with monoclonal gammopathies (2). There are no official guidelines for the optimal approach to screening patients for the presence of an abnormal monoclonal immunoglobulin, but a consensus has developed that a combination of tests is probably needed (3).
Enter mass spectrometry (MS). Many researchers have used this sensitive and specific technology for some time to analyze a variety of clinically important serum proteins, usually relying on the detection of a specific peptide after protease digestion of the original sample. Over a decade ago, investigators used this approach to analyze monoclonal light chains found in patients with primary amyloidosis. Recently, a group of clinicians and scientists at Mayo Clinic have extended these studies, using MS to study light chains in multiple myeloma and MGUS. Initially they took the traditional approach, using tryptic peptides specific for the unique variable region of monoclonal light chains ("clonotypes") as a new way to monitor myeloma patients after treatment (4). But these investigators soon hit upon a much more interesting (and more practical) approach: using MS to directly evaluate the heterogeneity of the patient's serum immunoglobulin in exactly the same way that protein electrophoresis does.
Just as each clonal plasma cell produces an electrophoretically homogeneous immunoglobulin, it appears that each also produces an immunoglobulin with a unique mass-to-charge (m/z) ratio when analyzed by MS. And, just as a spike appears when SPE or CZE in a patient with myeloma is examined, a similar spike appears when the patient's m/z spectrum is compared with that of normal pooled serum.
The group at Mayo Clinic employed antibodies to k and A light chains to capture and concentrate the patient's immunoglobulin. After reducing the disulfide bonds holding the heavy and light chains together, they were able to identify unique light chains derived from monoclonal immunoglobulins by using liquid chromatography coupled with electrospray ionization (ESI) and time of flight (TOF). Their initial reports included proof-of-concept (using a therapeutic monoclonal antibody spiked into normal serum) and detection of minimal residual disease in treated myeloma patients (5). Subsequently, the investigators switched to MALDI rather than ESI in an attempt to enhance throughput and develop an approach that could be adopted as a routine clinical procedure (6). In this issue of Clinical Chemistry, these investigators report their attempt to use MALDITOF MS as a clinical test to screen for the presence of M-components, identify the heavy and light chain, and quantify the concentration of intact monoclonal immunoglobulin.
MALDI-TOF MS was evaluated as a screen in two different ways (and each is reported separately). In one report (7), the group captured immunoglobulins by using antisera to [kappa] and [lambda] light chains, and then reduced disulfide bonds and scanned for light chains with restricted mlz ratios. They selected a cohort of samples of which approximately 75% were positive for an abnormal monoclonal immunoglobulin by conventional testing. These included samples in which the M-component was obvious (positive by SPE) as well as subtle (negative by SPE but positive by IFE). The results obtained with their MALDI-TOF MS assay (which they call "MASSSCREEN") perfectly matched the results obtained using SPE. However, there were discrepancies when their MS results were compared with IFE. Because the authors did not have access to detailed clinical information about these patients, the significance of these discrepancies is unclear.
In the second report (8), they describe a MALDITOF MS version of immunofixation (which they amusingly call "MASS-FIX"). Immunoglobulin is separately captured by antisera to the heavy chains of IgG, IgA, and IgM, as well as [kappa] and [lambda], and then the m/z distributions of the mass spectra (this time for both heavy and light chains) are examined separately after reducing disulfide bonds. MS analysis matched IFE almost perfectly both in terms of detecting M-components and identifying the heavy and light chains. This second report also includes information about how MS might be used (in conjunction with other analyses) to quantify the concentration of intact monoclonal immunoglobulin, an important result that conventional "fixation" cannot provide.
To be sure, there are still some issues that need to be addressed before laboratories replace their electrophoresis equipment with MALDI-TOF MS analyzers. In the first report, unblinded readers did very well but blinded readers, unfamiliar with MS, did less well. This is probably not unexpected, and laboratorians who review SPE, CZE, and IFE results should certainly understand the problems associated with small peaks and interference from other serum proteins. Also, quantification using MS may need more refinement. The authors relied on the proportion of the abnormal light chain to calculate the concentration of the intact monoclonal immunoglobulin based on the total IgG using nephelometry. Using the mass spectra of isolated heavy chains is challenging due to variable glycosylation as well as poor ionization relative to the light chains. Because the spectra of the normal polyclonal background may overlap small peaks using MS, some of the problems that currently plague M-component quantification when this occurs using SPE or CZE may still be a problem.
It also appears that serum FLCR may still have a role even if MALDI-TOF MS replaces electrophoresis because, in the first report, a significant number of samples that were negative by MALDI-TOF MS did have abnormal serum FLCR, indicating that this new approach is not yet ready to be a single test screen for monoclonal gammopathy. The authors did not include serum FLCR in their second study. Interestingly, the investigators at Mayo Clinic are already on their way to addressing this using MS. By omitting the reducing step after capturing immunoglobulins with antisera to [kappa] and [lambda] light chains, they have essentially developed an MS version of the serum free light chain immunoassay, made specific for monoclonal free light chain by visual inspection of the mass spectra (9).
Clearly, this is an exciting new way for the laboratory to help diagnose and manage multiple myeloma and related disorders. Combining multiple approaches to detection using one instrument may soon produce a single test screen for MGUS. And a more sensitive and specific way to monitor therapy (especially as therapeutic monoclonal antibodies become more widely used) will help patients avoid relapse.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
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James D. Faix  *
 Department of Pathology, Montefiore Medical Center, Bronx, NY.
* Address correspondence to the author at: Core Laboratory Administrative Office, 111 East 210th St., Foreman 8--Silver Zone, Bronx, NY 10467. E-mail: email@example.com. Received July 27, 2016; accepted July 28, 2016.
Previously published online at DOI: 10.1373/clinchem.2016.261933
 Nonstandard abbreviations: MGUS, monoclonal gammopathies of undetermined significance; SPE, serum protein electrophoresis; CZE, capillary zone electrophoresis; IFE, Immunofixation electrophoresis; FLCR, free light chain ratio; MS, mass spectrometry; TOF, time of flight; ESI, electrospray ionization.