Bence Jones proteins are monoclonal FLC that are detected in concentrated urine using PEL and IFE. These urinary FLCs are low molecular weight peptides that are removed from serum by the kidneys and are either metabolized or excreted in the urine. An alternative strategy to detect and quantitate the Bence Jones proteins would be to directly quantitate the FLC in serum. Early approaches used the size difference between intact immunoglobulins and FLC to separate the molecules, and then quantitated the FLC with light chain immunoassays. These multistep procedures never migrated into clinical laboratories. Subsequent approaches were based on using antisera that were specific for immunoglobulin light chain epitopes that were obscured when the light chain peptides were bound to the heavy chain peptides (Fig. 25.2). The specificity for these “cryptic” light chain epitopes meant that FLC could be accurately quantitated even in the presence of the large concentrations of intact immunoglobulin found in serum. The introduction of automated assays for the quantitation of immunoglobulin FLC has given clinical laboratories a new tool for evaluating AL. The FLC assays have increased the diagnostic sensitivity for identification of light chain diseases such as AL, and in addition they have improved disease monitoring and prognosis. The FLC assay was described by Bradwell and colleagues in 2001 [10]. The method is an automated nephelometric assay that uses a commercially available reagent set of polyclonal antibodies to quantitate both κ FLC and λ FLC by immunonephelometry (FREELITE™: The Binding Site, San Diego, Calif.). The antibodies show no reactivity by IFE or by Western blots to light chains contained in intact immunoglobulin. Sensitive hemagglutination assays showed reactivity to the appropriate FLC at dilutions above 1:16,000 and no reactivity to light chains contained within intact Ig at antisera dilutions >1:2. The assay reagents therefore appear to have a minimum of a 10,000-fold difference in reactivity to FLC compared to light chain contained within intact Ig. This high specificity allows κ and λ FLC to be quantitated in the presence of a large excess of serum IgG, IgA, and IgM. Similar to the quantitation of elevated immunoglobulins, elevations in FLC concentrations do not indicate monoclonality. The relationship between κ and λ light chain secretion, however, is predictable in normal plasma cell populations. The ratio of κ to λ light chain synthesis is approximately 2:1. Significant deviations from this ratio suggest clonal light chain synthesis. If the ratio of κ to λ FLC (rFLC) is significantly greater than normal, it suggests clonal proliferation of a κ-producing plasma cell. If the ratio is below normal, it suggests clonal excess of λ FLC.

A reference range study was performed with sera from healthy donors aged 21–90 years [11] whose sera were screened by PEL and IFE to exclude samples with a monoclonal protein [e.g., unknown MGUS patients]. The κ FLC and λ FLC concentrations were assessed, and the rFLC was calculated. This reference range study revealed an apparent effect of age on the 2 FLC concentrations, but the rFLC showed no age dependence. Cystatin C (a marker of renal clearance) showed the same apparent age dependence as the 2 FLC concentrations. The increase in serum FLC concentrations with age is most likely due to reduced renal clearance, and the use of the rFLC ratio normalizes the effects of reduced clearance.

Concentrations of κ and λ FLC may be abnormal due to immune suppression, immune stimulation, reduced renal clearance, or monoclonal plasma cell proliferative diseases. Serum from patients with either polyclonal hypergammaglobulinemia or renal impairment for example, often have elevated κ FLC and λ FLC due to increased synthesis or reduced renal clearance, respectively. The rFLC, however, usually remains normal in these conditions [11]. A significantly abnormal rFLC should only be due to a plasma cell proliferative disorder that secretes excess clonal FLC and disturbs the normal balance between κ and λ secretion. An abnormally high ratio suggests expansion of a kappa-producing plasma cell clone, whereas an abnormally low ratio suggests expansion of a lambda-producing plasma cell clone. The κ and λ FLC reference ranges were defined as 95 % reference ranges, but a “diagnostic range” for the rFLC was defined by 100 % of the normal sample study (Table 25.2).

Recently, Barnidge et al. [12] described the application of a mass spectrometry (MS)-based platform routinely used by pharma for monoclonal therapeutic quality control, microLC-ESI-Q-TOF MS, for the rapid detection, quantification, and isotyping of monoclonal immunoglobulin in patient sera. Using reduced Ig-enriched serum, these authors utilized the mass distribution measurements to identify isotype and quantify the distribution of light chains. Figure 25.3 demonstrates the mass spectrum distribution in the presence and absence of a monoclonal protein. Limits of detection were significantly lower than existing serum measurements and the technique was able to detect residual M-protein in samples negative by PEL, IFE, and FLC. This methodology represents a major shift in the characterization of monoclonal immunoglobulins. Rather than using gel electrophoresis and anti-isotypic antibody reagents, the authors combine molecular mass and constant region fragment ions to create a more sensitive technique for detecting and monitoring monoclonal immunoglobulins.