Multiple sclerosis (MS) is the most common non-traumatic disabling disease affecting young adults [13]. Given its highly variable clinical course, an unmet need for objective diagnostic assessment in MS persists [14].

OCB detection reflects intrathecal B-cell activity, which are critical but nonspecific effector cells in MS. Nevertheless, the level of intrathecal B-cell activity could be an exciting field of research to separate MS from other CNS inflammatory diseases and target treatment. As OCB are a qualitative biomarker, their detection does not permit quantification of the intrathecal B-cell activity. Moreover, isoelectric focusing is a time-consuming procedure that requires an experienced biologist to provide reliable results [15]. Free light chains in the CSF may represent a quantitative tool to demonstrate intrathecal IgG [5]. In the last decade, FKLCs in the CSF have emerged as a new biomarker in MS [16]. However, a strong consensus on its role in MS is still lacking [6]. We examined a total of 50 individuals for CSF FKLC levels and related indices, who were categorized as MS, CIS, and OIND groups in addition to healthy control group.

MS vs. healthy control and OIND groups. Patients with MS showed significantly elevated CSF FKLC levels as compared to healthy control and OIND groups. With regards to FKLC indices, MS group has also presented significantly higher values for all indices vs. controls and OIND. Our results confirm with previous results described by Zeman et al. in 2016 [17], Crespi et al. in 2017 [18], Senel et al. in 2019 [5], Duell et al. 2020 [11]. Ferraro et al. in 2020 [8], and Levraut et al. in 2023 [15]. With this regards, Machado-Santos et al. 2018 [19] have reported the presence of evidences that CNS B-cell activity is higher in MS than in other autoimmune disorders, as pointed out by pathologic analyses, showing a substantial perivascular B-cell infiltrate in MS compared with other inflammatory diseases [16]. Levraut et al. in 2023 have also reported that the high level of FKLC and indices in patient group vs. disease control group is expected, based on the growing evidence that CNS B cell activity is increased in MS with increased intrathecal synthesis of FKLC [15].

Comparing MS to OIND patients as regards the established markers of the work-up of MS: the IgG index and IEF for CSF–OCB, only the OCB was more prevalent among MS cases. Meanwhile, the IgG index has shown no significant difference, in contrast to CSF–FKLC and indices, as mentioned before. A finding which might point to a possible superior clinical impact of FKLC metrics over IgG index in MS diagnosis vs. other mimics. Our results are corroborated by other authors Crespi et al. who demonstrated that FKLC index turns out as superior to IgG index concerning sensitivity, positive and negative predictive values in MS diagnosis. Nevertheless, this point still warrants further studies on larger scales [18, 20].

CIS vs. OIND group. OCBs offer prognostic information concerning the development of MS after a first clinical suggestive event, the CIS. In these cases, detection of OCBs is clinically relevant and can help to identify patients with a high risk of future relapses [21]. Therefore, among our studied groups, CIS cases were also compared vs. OINDs for FKLC metrics as well as IgG index. CSF–FKLC levels and the calculated indices were all significantly higher in CIS cases than OIND patients, which goes in line with other studies [5, 15]. In contrast to FKLC metrics, IgG index failed to reveal any significant difference between our CIS and OIND groups. Similar to IgG index, the CSF–OCBs, which have shown no significantly increased prevalence in CIS compared to OIND. Our results are thus showing that FKLC metrics are still significantly differentiating CIS patients from those presenting with OINDs that could not be achieved by IgG index or OCB. Crespi et al. in 2019 reported that the FKLC index is more efficient than the IgG index as a quantitative test for intrathecal synthesis [17].

MS vs. CIS group. Viewing MS vs. CIS groups has demonstrated comparable results as regards FKLC indices but not the CSF–FKLC levels which were significantly higher among our MS than CIS cases. Partially similar to our results, are what have been reported by Leurs et al. in 2020 [3], and Levraut et al. in 2023 [15], who also found increased CSF–FKLC concentrations in MS than CIS patients, but in contrast to our study, they reported that the significant increase was also involving FKLC indices, which had also been reported in earlier studies by Senel et al. in 2014 [22], Presslauer et al. in 2016 [23], and Leurs et al. in 2020 [3], concerning the FKLC Q and KLC index. On the other hand, CSF–FKLC levels demonstrated comparable values between MS and CIS in a study published by Senel and co-authors in 2019 [5]. The difference between our results and other studies could be attributed to the small number included in our study, and could also be due to different methods used in assay of FKLC in different studies.

As regards the established lab markers in MS, OCB status showed higher percentage of positivity in our MS than CIS cases, however, IgG index was comparable between both groups [15] demonstrated a significantly higher percent of OCB positivity and significantly higher values of IgG index among their MS group than their CIS cases. The comparable values of IgG index in our study might be due to the limited number of cases included in the study, but, since it has been repeatedly reported that IgG index is known to be less sensitive in patients with MS than OCB [24], our finding is not assumed to be an unexpected one.

Healthy controls vs. OIND. The finding that FKLC index and FKLC IF have presented significantly higher values in OIND patients vs. healthy controls, excluding FKLC levels, we could point to superiority of indices against absolute values of FKLC in reflecting intrathecal immunoglobulin synthesis even in OINDs patients as they still maintain an ongoing intrathecal B cell activity, even if less pronounced than MS, nevertheless, is still higher than normal individuals. Our explanation is based on what has been reported in many reports as for example, Leurs et al. in 2020 who have reported that, similar as for the OCB, FKLCs indices can be elevated in inflammatory controls [3].

Diagnostic performance of CSF–FKLC metrics vs. IgG index and OCB. At best cutoff points differentiating MS from normal, diagnostic performances of CSF–FKLC and related indices were tested. FKLC biomarkers equally performed well as regards diagnostic sensitivity, specificity, NPV and PPV (100% each).

Studying diagnostic performance of OCB in differentiating MS from OINDs has shown a sensitivity and a NPV of 100% each, while the diagnostic specificity was 80% with a PPV of 95.5%. Various studies reported different OCBs specificities ranging from (82–94%) and sensitivities ranging from (82–98%) by Falip et al. in 2001 [25], Masjuan et al. in 2006 [26], Christiansen et al. in 2019 [27], and Ferraro et al. in 2020 [28], which are in line with our study, in the aspect that our study and others are all showing a relatively higher sensitivity and NPV than the specificity and the PPV of OCB as regards diagnosis of MS. The slightly lower specificity in our study than the reported range in the above-mentioned previous studies may be due to inclusion of OINDs as a disease control vs. MS, combined with another factor that is the limited number of patients included in this group. In support of this explanation, is a meta-analysis published in 2013, including 13,467 patients, which showed that the diagnostic specificity of OCB diminished if other inflammatory etiologies were considered [29].

With regard to FKLC and related indices at best cutoff points differentiating MS from OINDs, all parameters were presenting sensitivities, specificities, NPVs and PPVs of 100%. The meta-analysis study by Hegen et al. in 2022, has demonstrated that the FKLC IF had a diagnostic sensitivity ranging from 66 to 100% (average: 93%) and a specificity from 53 to 100% (average: 84%), which supports our result [6].

Moreover, we compared diagnostic performance of IgG index vs. FKLC metrics at best cutoff points to discriminate MS from OINDs, which has revealed comparable performance of both with regards to diagnostic specificities and PPVs (100%, each). In contrast, the diagnostic sensitivity and NPV of IgG index were significantly lower (52.4% and 33.3%, respectively) than those presented by FKLC metrics under study (100% each). Rosenstein and co-authors (2021) have reported better diagnostic specificity and sensitivity of FKLC index and FKLC IF than IgG index [30].

As shown in our study, the FKLC measures applied (CSF–FKLC concentration, FKLC index, FKLC IF and FKLC Q) show no difference in diagnostic accuracy. Multiple studies applied comparisons between different FKLC measures (CSF–FKLC concentration, FKLC IF and FKLC index). These studies have also shown similar diagnostic accuracy between all the used measures, however, as has been analysed by Hegen and colleagues, the statistical power for the comparison with the most employed studies was already less than 80% which yet cannot exclude the superiority of one over the other which requires further exploration [6].

Focusing on MS phenotype among our cases, RRMS presented with higher CSF–FKLC levels than PPMS, which was also higher in RRMS when compared to CIS. With regards to indices, FKLC index and FKLC IF were noticed to be higher in RRMS than PPMS but the difference did not reach a statistically significant level. A study by Rosenstein and colleagues 2021 [30] has reported a higher FKLC-index in RRMS than PPMS, however, statistical significance was borderline (p = 0.05). Moreover, a previous study by Levraut et al. in 2023 [15] has demonstrated that CSF FKLC were not statistically different between progressive MS and RRMS but both MS subgroups presented with higher CSF FKLC concentrations than CIS patients, and that RRMS presented with higher FKLC index and FKLC IF than progressive MS and CIS.