Diagnostic utility of kappa free light chains in multiple sclerosis

KEYWORDS:

• Free light chains
• dimers
• kappa
• lambda
• monomers
• multiple sclerosis

1. Introduction

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that leads to demyelination, axonal damage, and neuronal loss. There is no single test for MS: diagnosis of MS relies on the integration of clinical, imaging, and laboratory findings. The latest revisions of the McDonald criteria of 2017 for MS [1] have revitalized the role of cerebrospinal fluid (CSF) analysis in MS diagnostics. The evidence of an intrathecal immunoglobulin synthesis, i.e., presence of oligoclonal bands (OCB) in the CSF [2], is now being considered as a diagnostic criterion in MS.

Normally, immunoglobulin light chains are produced by B-cells in excess of heavy chains. While majority of light chains are bound to heavy chains to create complete immunoglobulins, small amounts of light chains remain unbound (‘free’) and are secreted as so-called free light chains (FLC). FLC exist as two isotypes (kappa or lambda), and each of them appears in two major molecular forms, namely, monomers (25 kDa) and dimers (50 kDa). Both kappa and lambda FLC are found in different bodily fluids, including serum, CSF, urine, saliva, tears, and synovial fluid. The physiological levels of FLC are low, but under various pathological conditions, these levels may be abnormally high.

In addition to oligoclonal immunoglobulin synthesis, the intrathecal production of FLC is currently also regarded as an important immunological response developing in the CNS of MS patients. The increased levels of FLC in the CSF of MS patients were first observed in the late 70s [3]. However, the early methods applied to study FLC were basically qualitative, quite laborious, and gained little attention in MS diagnostics. The current interest in FLC has been stimulated by recent achievements in the FLC quantification techniques, especially by the development of a highly sensitive nephelometric FLC assay.

2. Nephelometric FLC assay versus OCB test

Use of the nephelometric FLC assay has clearly demonstrated increased levels of kappa FLC in the CSF of MS patients [4–6]. Determination of FLC index was introduced and commonly used to account for blood-CSF barrier permeability (FLC CSF/serum ratio divided by albumin CSF/serum ratio) [7–9]. FLC index is thought to be more relevant than the mere concentration of FLC in CSF. These findings strongly suggest the diagnostic utility of the nephelometric FLC assay as a quantitative and technically simple method compared to the routinely used OCB test. However, different studies comparing sensitivity and specificity of the FLC assay to those of the OCB test obtained conflicting results [6,9–11]. Some authors have demonstrated superiority of FLC assay over the OCB test [10], or have shown that the FLC assay is more sensitive but less specific than OCB test [6]. These findings have been contradicted by recent studies which point to a higher sensitivity of OCB test [11] or to equal performance of both tests [9]. Controversial results have also been obtained in assessment of the prognostic value of kappa FLC levels in the conversion of clinically isolated syndrome (CIS) to MS [7,12,13].

Hence, there is no doubt that determination of kappa FLC level is helpful in the diagnosis of MS. However, keeping in mind the heterogeneous nature of MS disease, on the one hand, and the discrepancies in regard to diagnostic superiority of nephelometric FLC assay over the OCB test, on the other, it remains questionable whether determination of a single parameter (e.g., kappa FLC only) is diagnostically sufficient. In fact, a few nephelometric studies have pointed out that both kappa and lambda FLC levels, as well as their ratios, are important for precise diagnosis and prognosis of MS [14,15]. It has been proposed that combined use of the OCB test and the FLC assay could be more fruitful in diagnosing MS and that the kappa FLC level estimation may complement, but not replace the OCB test [9,11].

3. Limitations of nephelometric FLC assay

Although development and use of nephelometric FLC assay have opened new avenues in the studies of intrathecal immune response and offer a new approach in the diagnosis of MS, the pitfalls of this assay are now well recognized. In fact, antibodies used in the nephelometric FLC assay recognize only a limited number of epitopes (situated between heavy and light chains of intact immunoglobulins), and these antibodies may preferentially detect FLC dimers over monomers [16]. In addition, the two major commercial antibodies used in this assay, i.e. Freelite (The Binding Site Group Ltd., Birmingham, UK) and N Latex FLC (Siemens Healthcare Diagnostics Gmbh, Marburg, Germany) may provide different results for the same sample. According to a recent study [17], the observed quantitative discrepancies may arise be due to different immunoreactivities of these antibodies against FLC monomers and dimers, especially with respect to lambda FLC. Since the FLC levels measured using nephelometry represent the total sum of monomeric and dimeric FLC, certain degree of inaccuracy is inevitable in this test. These limitations of the FLC assay cannot be ignored in view of the CSF studies employing Western blot-based techniques [18–20], which showed that the abnormal immune response in MS involves not only increased FLC levels, but also changes in the FLC monomer/dimer ratios.

4. Electrophoretic methods of FLC analysis in MS

Compared to wide application of nephelometric assays, the number of studies employing electrophoretic analysis of FLC in the CSF is relatively small. These studies include immunodetection of FLC by using isoelectric focusing [21], agarose gel electrophoresis [22], or sodium dodecyl sulfate (SDS) electrophoresis [18,20–23]. While majority of the MS CSF samples tested by these methods have shown abnormally high levels of kappa FLC, the increased concentration of lambda FLC has also been invariably demonstrated in a part of MS patients. Furthermore, CSF-FLC analysis of MS patients, performed using a semiquantitative SDS electrophoresis-based Western blotting technique, has revealed three distinct pathological FLC monomer–dimer patterns typical of MS: (a) highly increased kappa CSF levels (both monomers and dimers) in absence of any significant increase in lambda FLC level (‘kappa MS type’);

(b) increased kappa FLC levels accompanied by abnormally high amounts of lambda FLC dimers (‘mixed kappa-lambda MS type’); (c) abnormally high level of lambda FLC dimers with normal levels of kappa FLC (‘lambda MS type’) [19]. These pathological patterns were absent in other non-MS neurological diseases, even when these cases showed abnormally increased total amount of FLC. Interestingly, it was found that the ‘lambda MS type’ was observed in 10% of adult MS patients [19] and in 30% of pediatric MS patients [20]. The sensitivity and specificity of this SDS electrophoresis-based diagnostic technique were higher than those of the OCB test performed using Sebia agarose gel electrophoresis coupled with immunofixation; however, final conclusions should be made after comparison with the isoelectrofocusing-based OCB analysis, which is still considered the ‘gold standard’ for demonstration of oligoclonal bands. In any case, the finding of increased levels of lambda FLC with a strong prevalence of dimers vs monomers [16–18,21] requires special attention, as it may be specifically related to MS and not to other neurological diseases ‘mimicking’ MS [18–20,23]. The obtained data emphasize the role of differential analysis of monomeric and dimeric FLC in the diagnosis of MS.

Taken together, the results obtained using electrophoretic techniques make it clear that

(a) analysis of both FLC subtypes, i.e., kappa and lambda, may significantly increase the accuracy of MS diagnosis, (b) high CSF levels of FLC per se may be indicative of but not sufficient for MS diagnosis, (c) molecular analysis of FLC forms accounting for the ratio of kappa and lambda monomers and dimers may be critical for accurate differentiation between MS and other neurological diseases ‘mimicking’ MS.

5. Concluding remarks

Since the above-mentioned methods require collection and examination of CSF, any attempts to develop less invasive techniques of FLC analysis should be welcomed. Actually, an increasing body of evidence suggests that in addition to the intrathecal immune response, the pathophysiology of MS involves activation of immune system outside the CNS. This point of view is supported by the reported changes in mucosal immunity of MS patients, including: increased number of immunoreactive cells in the saliva [24,25]; impairment of mucosal barrier of the gut [26]; presence of oligoclonal immunoglobulin bands and increased amounts of IgG and IgA in the tears [27–29]. Although these findings raise a hope that FLC analysis of the more easily accessible bodily fluids may be diagnostically useful in MS, this issue has been studied only by few authors and needs further confirmation and investigation [30,31].

Finally, comparison of different methodological approaches used to study FLC in MS shows that each of them comes with its own set of strengths and weaknesses. In contrast to the quantitative nephelometric FLC assay, the OCB test is a qualitative technique, highly dependent on skills and experience of laboratory personnel. The electrophoretic methods used to study FLC are laborious compared to nephelometric assays, but they provide diagnostically useful information which is not available by nephelometry. SDS electrophoresis-based Western blot technique allows separation of FLC from bound light chains, therefore, in contrast to the nephelometric assay, it makes possible the detection of FLC using the easily available commercial antibodies that recognize a wide spectrum of antigenic moieties of FLC, without differences in the reactivity between FLC monomers and dimers [32]. Understanding of the advantages and limitations of these techniques is essential for adequate interpretation of the obtained data. The question may be raised whether the combined use of different techniques of FLC analysis could be diagnostically useful. For example, the technically simple nephelometric assay may be the first to be used as a screening test to filter out the clear-cut cases with normal FLC levels which are believed to be incompatible with MS diagnosis. For patients showing abnormal levels of kappa or lambda FLC, the subsequent electrophoretic analysis of monomeric and dimeric FLC forms may be beneficial. Such approach may be especially helpful in cases where the results of routine clinical and paraclinical tests are equivocal; in these cases application of the reliable complementary diagnostic tests may contribute to precise diagnosis of MS.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewers disclosure

Peer reviewers on this manuscript have no relevant financial relationships or otherwise to disclose.

Additional information

Funding

This paper was not funded.