1134213 research-article20222022 MSJ0010.1177/13524585221134213Multiple Sclerosis JournalH Hegen, J Walde MULTIPLE SCLEROSIS MSJ JOURNAL Review Cerebrospinal fluid kappa free light chains for Multiple Sclerosis Journal 2023, Vol. 29(2) 169­–181 the diagnosis of multiple sclerosis: A systematic DOI: 10.1177/ https://doi.org/10.1177/13524585221134213 13524585221134213 https://doi.org/10.1177/13524585221134213 review and meta-analysis © The Author(s), 2022. Article reuse guidelines: sagepub.com/journals- Harald Hegen, Janette Walde , Klaus Berek , Georgina Arrambide , permissions Sharmilee Gnanapavan , Batia Kaplan, Michael Khalil , Ruba Saadeh, Charlotte Teunissen , Hayrettin Tumani, Luisa M Villar, Maria Alice V Willrich, Henrik Zetterberg and Florian Deisenhammer Abstract Correspondence to: H Hegen Background: Intrathecal immunoglobulin-G synthesis is a hallmark of multiple sclerosis (MS), which Department of Neurology, can be detected by oligoclonal IgG bands (OCB) or by κ-free light chains (κ-FLC) in cerebrospinal fluid. Medical University of Innsbruck, Anichstrasse 35, Objective: To perform a systematic review and meta-analysis to evaluate whether κ-FLC index has simi- Innsbruck A-6020, Austria. lar diagnostic value to identify patients with clinically isolated syndrome (CIS) or MS compared to OCB, harald.hegen@i-med.ac.at Harald Hegen and to determine κ-FLC index cut-off. Klaus Berek Methods: PubMed was searched for studies that assessed diagnostic sensitivity and specificity of κ-FLC Florian Deisenhammer Department of Neurology, index and OCB to discriminate CIS/MS patients from control subjects. Two reviewers following pre- Medical University of Innsbruck, Innsbruck, Austria ferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines performed study Janette Walde eligibility assessment and data extraction. Findings from studies were analyzed with bivariate mixed Department of Statistics, models. Faculty of Economics and Statistics, University of Results: A total of 32 studies were included in the meta-analysis to evaluate diagnostic value of κ-FLC Innsbruck, Innsbruck, Austria index. Sensitivity and specificity ranged from 52% to 100% (weighted average: 88%) and 69% to 100% Georgina Arrambide Centre d’Esclerosi (89%) for κ-FLC index and from 37% to 100% (85%) and 74% to 100% (92%) for OCB. Mean difference Múltiple de Catalunya, of sensitivity and specificity between κ-FLC index and OCB was 2 and −4 percentage points. Diagnostic Department of Neurology/ Neuroimmunology, Hospital accuracy determined by mixed models revealed no significant difference between κ-FLC index and OCB. Universitari Vall d’Hebron, Universitat Autónoma de A discriminatory cut-off for κ-FLC index was determined at 6.1. Barcelona, Barcelona, Spain Conclusion: The findings indicate that κ-FLC index has similar diagnostic accuracy in MS as OCB. Sharmilee Gnanapavan Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of Keywords: Cerebrospinal fluid, kappa free light chains, multiple sclerosis, clinically isolated syndrome, London, London, UK diagnosis, biomarker, index, intrathecal fraction, meta-analysis, systematic review Batia Kaplan Laboratory of Hematology, Sheba Medical Center, Ramat Date received: 23 June 2022; revised: 9 September 2022; accepted: 4 October 2022 Gan, Israel Michael Khalil Department of Neurology, Medical University of Graz, Graz, Austria Introduction In the last decade, κ-free light chains (κ-FLC) in the Ruba Saadeh Cerebrospinal fluid (CSF) analysis is of high impor- CSF have emerged as new biomarker in MS. κ-FLC Department of Laboratory tance in the diagnostic work-up of patients with sus- are secreted by B cells along with intact immuno- Medicine and Pathology and Department of Neurology, pected multiple sclerosis (MS).1 Evidence of globulins and accumulate in the CSF in case of Mayo Clinic, Rochester, intrathecal immunoglobulin G (IgG) synthesis in the chronic intrathecal inflammation.5 In contrast to MN, USA CSF, although not specific for MS, substitutes for dis- OCB, determination of κ-FLC has considerable Charlotte Teunissen Neurochemistry Laboratory, semination in time according to current diagnostic advantages. First, κ-FLC are measured by neph- Department of Clinical criteria2 and increases diagnostic certainty in the elometry or turbidimetry, which are easy, reliable, Chemistry, Amsterdam Neuroscience, Vrije appropriate clinical setting.3 Currently, the gold stand- labor-saving, and cost-effective methods. Second, Universiteit, Amsterdam ard to prove intrathecal IgG synthesis is the detection the determination of κ-FLC returns a metric and UMC, Amsterdam, The Netherlands of CSF-restricted oligoclonal IgG bands (OCB).4 rater-independent result.6,7 journals.sagepub.com/home/msj 169 Multiple Sclerosis Journal 29(2) Hayrettin Tumani CSF Laboratory, Department Most studies used the κ-FLC index to prove an Patients of any age were included, with no restrictions of Neurology, University of intrathecal synthesis and showed its high diagnostic on MS disease course, disease duration, disability, Ulm, Ulm, Germany accuracy to discriminate patients with MS from other comorbidities or treatment. Diagnosis of CIS or MS Luisa M Villar Immunology Department, neurological diseases.8–12 However, a strong consen- should be stated with referring to the established diag- Hospital Universitario sus on the role of κ-FLC as biomarker in MS is still nostic criteria.2,14–16 Only studies using immunoneph- Ramón y Cajal, Madrid, Spain lacking. This might be due to heterogeneity between elometry or immunoturbidimetry to determine κ-FLC Maria Alice V Willrich published studies ranging from different patient popu- concentrations in paired CSF and serum/plasma sam- Department of Laboratory lations included, different assays used, to the different ples, or in the CSF only were included. When patient Medicine and Pathology, Mayo Clinic, Rochester, κ-FLC measures (e.g. κ-FLC index versus absolute populations overlapped in several articles, only the MN, USA CSF κ-FLC concentration) and cut-off values applied. one with the most complete information was included. Henrik Zetterberg Studies could be retrospective or prospective. Department of Psychiatry and Neurochemistry, Therefore, we aimed to compare the diagnostic value Institute of Neuroscience of κ-FLC index to OCB in patients with clinically iso- and Physiology, Sahlgrenska Academy, University of lated syndrome (CIS) and MS and to identify an Data extraction Gothenburg, Mölndal, appropriate cut-off for κ-FLC index. Furthermore, we Data extraction forms were created. Data were Sweden/Clinical Neurochemistry Laboratory, aimed to elucidate differences to other κ-FLC extracted from selected articles independently in Sahlgrenska University measures. duplicate (HH and FD). Disagreements were resolved Hospital, Mölndal, Sweden/Department of by consensus and if needed with another author (JW). Neurodegenerative Disease, Queen Square Institute of Neurology, University Methods The following data were extracted: the first author, College London, London, This study followed the preferred reporting items for publication year, number of patients per disease group UK/UK Dementia Research Institute, University College systematic reviews and meta-analyses (PRISMA) (i.e. CIS or MS, control group), type of samples col- London, London, UK/ reporting guideline.13 lected (CSF, serum, or plasma), method used for κ- Hong Kong Center for Neurodegenerative Diseases, FLC detection (principal method (nephelometry, Hong Kong, China turbidimetry), assay kit (Freelite, N Latex), and the Search strategy platform, as appropriate), diagnosis and used diagnos- A comprehensive search of the electronic database tic criteria of CIS/ MS patients, allocation of controls PUBMED was performed on 1 February 2022. The to one of predefined control groups (non-inflamma- search included the following terms: “free light tory neurological disease control (NINDC), inflam- chain” and “multiple sclerosis.” “Multiple sclero- matory neurological disease control (INDC), sis” was searched as a MeSH Term and keyword, peripheral inflammatory neurological disease control “free light chain” was searched as keyword. The (PINDC), symptomatic control (SC), healthy control publication date was restricted from 1 January 2000 (HC),17 and non-neurological disease control (prior to that date there were no studies on κ-FLC (NNDC)), corticosteroid treatment prior to sample in CSF as determined by nephelometry or turbidim- collection in CIS/ MS patients, disease-modifying etry) to 1 February 2022. Only original articles in treatment at the time of sample collection in CIS/MS English were included. Two authors (HH and FD) patients, number of positive OCB test results (pattern independently conducted the literature search, that II or pattern III)4 in the CIS/MS patients, number of is, screened titles and abstracts of identified articles negative OCB test results in the control subjects, after removing duplicates, then independently number of positive test results for κ-FLC index, assessed the full text of potentially relevant articles IFκ-FLC, CSF κ-FLC concentration or Qκ-FLC in the for inclusion and exclusion criteria. Discrepancies CIS/MS patients, number of negative test results for between the two authors were discussed and κ-FLC index, IFκ-FLC, CSF κ-FLC concentration or resolved. Qκ-FLC in the control subjects, the applied cut-off val- ues to define test positivity. If the number of posi- tively or negatively tested patients and controls, Selection criteria respectively, was not available, the reported diagnos- Studies were included if they were original articles tic sensitivity and specificity were used to back-calcu- investigating the diagnostic value of κ-FLC index, the late this number. percentage intrathecal κ-FLC fraction (IFκ-FLC), CSF κ-FLC concentration or κ-FLC quotient (Qκ-FLC) in patients with CIS or MS compared to any healthy or Statistical analysis disease control. Definition and calculation of κ-FLC Studies with data available of diagnostic sensitivity index, IFκ-FLC or Qκ-FLC are provided in the supple- and specificity of κ-FLC index, IFκ-FLC, CSF κ-FLC mental material. concentration or Qκ-FLC to discriminate CIS or MS 170 journals.sagepub.com/home/msj H Hegen, J Walde et al. patients from controls were included in the quantita- Results tive meta-analysis. The search strategy identified 234 references (Figure 1). After removing duplicate records, 101 references Both sensitivity and specificity of each κ-FLC meas- were screened for potential relevance through titles ure were compared to sensitivity and specificity of and abstracts. This process yielded 66 potentially eli- OCB used within the same study thereby holding the gible studies that underwent full-text eligibility within study conditions for both parameters constant review. Of these, 38 studies were included in the sys- (e.g. characteristics of CIS/MS patients and control tematic review.8–12,24–57 Thirty-two studies addressed subjects, administration of prior immune treatment). the diagnostic value of κ-FLC index, 13 studies of Findings are presented in forest plots separately for IFκ-FLC, 10 studies of CSF κ-FLC concentration and 3 sensitivity and specificity. The magnitude of hetero- studies of Qκ-FLC; 15 studies addressed more than one geneity was assessed by Higgins/Thompson’s I2, of these parameters. which is an estimate of the variability across studies based on heterogeneity rather than chance. I2 ranges from 0% to 100%; and low, moderate, and high het- κ-FLC index versus OCB erogeneity are indicated by I2 values below 25%, A total of 32 studies addressed the diagnostic accu- 50%, and 75%, respectively.18 racy of κ-FLC index including 3322 patients with CIS/ MS and 5849 controls. All studies reported sig- To consider simultaneously within-study variation, nificantly elevated κ-FLC index in CIS/ MS patients between-study variation and the degree of correlation compared to controls. Diagnostic sensitivity and between sensitivity and specificity because of the cho- specificity ranged from 52% to 100% (weighted aver- sen cut-off point, a bivariate mixed model was age: 88%) and 69% to 100% (89%) for κ-FLC index employed.19 Using REML (restricted maximum likeli- and from 37% to 100% (85%) and 74% to 100% hood) for estimation, the estimates of sensitivity and (92%) for OCB. specificity and their 95% elliptical confidence interval (CI) were used to compare the accuracy of each κ- FLC measure with OCB. To ensure the validity of our Studies differed with regard to demographics, clinical meta-analysis, we did an outlier diagnostic.20 The esti- characteristics, and laboratory methods. While 22 mated bivariate distribution was used to show sum- studies included distinct cohorts of MS patients and 8 mary receiver operating curves (sROC). The findings studies included patients with CIS, 8 studies analyzed were checked for robustness by splitting the studies mixed cohorts comprising both patients with CIS and according to their different patients and control groups MS. Twenty-four (75%) of 32 studies applied either and performing the corresponding sub-analyses. the 2010 or 2017 revised McDonald criteria in CIS/ MS patients, 3 studies used earlier diagnostic criteria, A power analysis was conducted21 to investigate and 5 studies did not specify the applied criteria. whether sample size was sufficient to interpret statis- Nephelometry was applied in 22 (69%) studies and tically non-significant findings. A significance level turbidimetry in 9 (28%) studies; 16 (50%) studies of 5% and the number of studies included in the meta- used the Freelite assay, and 15 studies (47%) the N analysis were used. A large between-study heteroge- Latex assay. One study (3%) applied different type of neity was assumed. A difference in sensitivity and platform and assay in the patient and control group. specificity of 5 percentage points (pp) was regarded Cut-off values of the κ-FLC index denoting test posi- as substantial. tivity ranged from 2.4 to 20.0. For further details on each study characteristics, we refer to Supplemental Cut-off values for the discrimination between CIS/ Table S1. MS patients and control subjects were determined for the κ-FLC index and the CSF κ-FLC concentration. Bivariate confidence intervals of sensitivity and spec- First, we performed power analysis for a bivariate ificity for each of these two κ-FLC measure were mixed model to ensure a valid interpretation for not computed at the 99% confidence levels. The weighted statistically significant differences. For that, we used average over all cut-offs from the studies in this con- a significance level of 5%, a sample size of 32 studies, fidence interval was calculated. The weighting was the studies within variance, assumed a large between based on the sample size of the studies. heterogeneity and chose a 5 pp difference in sensitiv- ity or specificity between κ-FLC index and OCB as A two-sided significance level of 5% was considered important to detect (e.g. OCB 90% and κ-FLC index statistically significant. R software22 and the package 85%). Therewith, we obtained a power of 98.7% for mada23 were used for all analyses. sensitivity and 99.9% for specificity. journals.sagepub.com/home/msj 171 Multiple Sclerosis Journal 29(2) Figure 1. PRISMA flow diagram of study identification, screening, eligibility review, and selection for this systematic review and meta-analysis. PRISMA: preferred reporting items for systematic reviews and meta-analyses; CSF: cerebrospinal fluid; FLC: free light chain; MS: multiple sclerosis. Forest plots were used to visualize sensitivities and also of OCB. This implies that not the type of assay, specificities and to get an overview of between-study but another confounding factor is responsible for this heterogeneity. They showed mostly overlapping con- observation. Indeed, studies using the Freelite assay fidence intervals and revealed low to moderate included more frequently patients with CIS (5 of 13 between-study heterogeneity (I2 = 29.5%; (95% CI): studies), while studies using the N Latex assay were 0, 55.0%; Figure 2). done with MS patients mainly (8 of 10 studies). The bivariate model analyzing the diagnostic accuracy of Mean difference of diagnostic sensitivity between κ- κ-FLC index and OCB controlling for the type of dis- FLC index and OCB was 2 pp and −4 pp of specific- ease (CIS vs MS) confirmed that patients with CIS ity. The estimated bivariate mixed model assessed no showed a lower sensitivity than patients with MS for statistically significant difference between κ-FLC the κ-FLC index, but also for OCB (Supplemental index and OCB for the accuracy to discriminate CIS Table S8). and MS patients from controls (Figure 3, Supplemental Table S5). In addition, we evaluated a possible impact In analogy, we investigated the possible impact of the of the type of assay on the diagnostic sensitivity and platform (nephelometry or turbidimetry) on the diag- specificity of κ-FLC index and observed a statisti- nostic sensitivity and specificity of κ-FLC index and cally significant lower sensitivity with the Freelite observed at first a statistically significantly lower sen- assay (p < 0.001, Supplemental Table S6). Further sitivity for turbidimetry (Supplemental Table S9). In analysis comparing the accuracy of κ-FLC index and the subgroup turbidimetry as well as in the subgroup OCB controlling for the type of assay and excluding nephelometry, the sensitivity of κ-FLC index and mixed cohorts of CIS/ MS patients (Supplemental OCB did not significantly differ; thus, a potential Table S7) showed that in the group of Freelite assay impact of the platform could be excluded not only sensitivity of κ-FLC index was lower, but (Supplemental Table S10). 172 journals.sagepub.com/home/msj H Hegen, J Walde et al. Figure 2. Forest plot of studies comparing the diagnostic accuracy of κ-FLC index and OCB. In the left column, forest plot of sensitivities for the studies included in the meta-analysis are shown for κ-FLC index (above) and OCB (below); in the right column forest plot of specificities for the studies included in the meta-analysis for κ-FLC index (above) and OCB (below) are provided. Confidence intervals are computed at a 95% confidence level. FLC: free light chain; OCB: oligoclonal band. journals.sagepub.com/home/msj 173 Multiple Sclerosis Journal 29(2) Figure 3. Comparison of the diagnostic accuracy of κ-FLC index with OCB to identify CIS/MS patients Bivariate summary estimates of sensitivity and specificity for κ-FLC index with OCB and the corresponding 95% confidence ellipse around these mean values are shown as well as the original data of the meta-analysis together with the corresponding sROC curves. FLC: free light chain; OCB: oligoclonal band; sROC: summary receiver operating curve; CIS: clinically isolated syndrome; MS: multiple sclerosis. To further investigate the impact of different patient IFκ-FLC in patients with CIS/MS compared to controls. (MS, CIS, mixed CIS/MS) and control groups (non- IFκ-FLC showed a diagnostic sensitivity ranging from inflammatory diseases, inflammatory and/or non- 66% to 100% (weighted average: 93%) and a speci- inflammatory diseases), subgroup analyses were ficity from 53% to 100% (84%). In comparison, OCB performed. This robustness check revealed consistent had a diagnostic sensitivity of 57%–97% (89%) and a findings for all subgroups (Supplemental Figures S1 specificity of 74%–100% (91%). and S2). Study characteristics concerning demographics, clini- A cut-off for κ-FLC index at 6.1 was determined to dis- cal variables, and laboratory methods are detailed in criminate CIS/MS patients from controls (Supplemental Supplemental Table S2. A total of nine studies Figure S3). included MS patients, three studies CIS patients, and four studies analyzed mixed cohorts comprising both CIS and MS patients. Eleven (85%) of 13 studies Intrathecal κ-FLC fraction versus OCB applied either the 2010 or 2017 revised McDonald The diagnostic accuracy of IFκ-FLC was addressed by criteria in CIS/MS patients. Nephelometry was used 13 studies including 1428 CIS/MS patients and 3299 in 11 (85%) studies, turbidimetry in one (8%) study. controls. All studies reported significantly elevated Four (31%) studies used the Freelite assay, while 174 journals.sagepub.com/home/msj H Hegen, J Walde et al. eight studies (62%) the N Latex assay. One study Mean difference of diagnostic sensitivity between applied different type of platform and assay in the CSF κ-FLC index and OCB was 0 pp and of specific- patient and control group. Studies applied different ity −3 pp. Diagnostic accuracy between CSF κ-FLC formulae for the definition of the cut-off (i.e. the Qlim concentration and OCB to discriminate CIS/MS κ-FLC): six (46%) studies applied the formula by Reiber patients from controls was similar (Supplemental et al.,46 five by Presslauer et al.,58 and one study by Figure S7). A cut-off for CSF κ-FLC concentration of Senel et al.11 0.96 mg/L to discriminate CIS/MS patients from con- trols was observed (Supplemental Figure S8). Forest plots of sensitivity and specificity are shown in However, the calculated power was smaller than 80% Supplemental Figure S4 and revealed a high between- due to the small number of studies. study homogeneity (I2 = 5.9%; (95% CI): 0, 57.8%). Diagnostic sensitivity between IFκ-FLC and OCB dif- κ-FLC quotient versus OCB fered on average by 4 pp and specificity by −8 pp. The Two studies including MS patients and one study with diagnostic accuracy as determined by the mixed a cohort of CIS patients investigated the diagnostic model revealed no difference between IFκ-FLC and accuracy of Qκ-FLC. These studies included a total of OCB to discriminate CIS and MS patients from con- 256 CIS/MS patients and 1249 controls. Study char- trols (Supplemental Figure S5). We also considered acteristics are given in Supplemental Table S4. different formulae (Presslauer versus Reiber formula) Overall, sensitivity of Qκ-FLC ranged from 92% to in the model, but did not find evidence for an impact 94% (weighted average: 93%) and specificity from on diagnostic sensitivity and specificity. However, the 73% to 96% (95%), while OCB showed a sensitivity calculated power for the model was smaller than 80% of 91% to 100% (96%) and a specificity of 93% to due to the small number of studies. 100% (94%) in these studies. Mean difference of diagnostic sensitivity between Qκ-FLC index and OCB was 3 pp and of specificity −8 pp. Forest plots of sen- CSF κ-FLC concentration versus OCB sitivity and specificity are shown in Supplemental A total of 10 studies addressed the value of CSF κ- Figure S9. FLC including 901 patients with CIS/MS and 2251 controls. All studies reported significantly elevated CSF κ-FLC concentration in patients with MS com- Comparisons between different κ-FLC measures pared to controls. CSF κ-FLC concentration showed a Studies that applied different κ-FLC measures on the diagnostic sensitivity ranging from 66% to 96% same patient cohort were eligible: 7 studies compared (weighted average: 84%) and a specificity from 70% κ-FLC index with CSF κ-FLC concentration, 4 stud- to 100% (87%). In comparison, OCB had a diagnostic ies IFκ-FLC with CSF κ-FLC concentration, and 11 sensitivity of 57% to 100% (86%) and a specificity of studies compared κ-FLC index with IFκ-FLC. 72% to 100% (88%). Diagnostic accuracy between all three κ-FLC meas- ures was similar; however, the statistical power for Seven studies included distinct groups of MS the comparison with the most employed studies was patients, three studies patients with CIS, while three already less than 80% (Supplemental Figure S10). studies analyzed mixed cohorts. In all studies, either the 2010 or 2017 revised McDonald criteria were applied for CIS/MS patients. Nephelometry was Discussion used in eight (80%) studies and turbidimetry in the This systematic review and meta-analysis provides remaining two (20%) studies; half of the studies evidence that the determination of intrathecal κ-FLC used the Freelite assay, whereas the other half the N shows a high diagnostic accuracy to discriminate Latex assay. Cut-off values for the CSF κ-FLC con- patients with CIS and MS from other neurological centration test positivity ranged from 0.3 to 7.1 mg/L. diseases. All approaches to capture intrathecal κ- Detailed study characteristics are shown in FLC—including the κ-FLC index, the IFκ-FLC, Supplemental Table S3. the Qκ-FLC, and the absolute CSF κ-FLC concentra- tion—showed comparable performance, which was Forest plots of sensitivity and specificity are provided equal to OCB testing. With high statistical power of in Supplemental Figure S6. They show a low to mod- 99%, significant evidence exists just for κ-FLC index erate between-study heterogeneity (I2 = 28.7%; (95% with 32 studies performed on approximately 3300 CI): 0, 63.2%). CIS/MS patients and 5800 control subjects. journals.sagepub.com/home/msj 175 Multiple Sclerosis Journal 29(2) κ-FLC in the CSF—similar to immunoglobulins or been published. In general, different cut-off values other proteins—originate either from blood by diffu- might apply depending on the clinical question, for sion across the blood–CSF barrier, or are produced example, to provide an upper reference limit deter- within the intrathecal compartment under pathologi- mined in a (non-inflammatory) control population17 cal conditions.59 Conceptually, it seems necessary to or to differentiate MS from other INDC. Furthermore, determine the locally synthesized κ-FLC fraction cut-off values might vary whether the main aim is to separate from the blood-derived fraction (as it is also increase diagnostic sensitivity or specificity.63 Here, done for IgG) to prove intrathecal B cell activity. we observed a discriminatory cut-off for κ-FLC index Therefore, majority of studies used the κ-FLC index8– at 6.1 to differentiate CIS/MS patients from controls, 12,24–45,47–49,51,52 or the IF κ-FLC. 9–11,34–37,39,42,50,52–54 Both as well as at 0.96 mg/L for CSF κ-FLC concentration. approaches consider the albumin quotient (Qalb) Even though the cut-off for κ-FLC index8,9,12,30 as which is an established marker of the blood–CSF- well as for the CSF κ-FLC concentration30,55 is in line barrier function60 and correct for the absolute serum with those identified by several large—partly multi- κ-FLC concentration. Few studies used the Qκ- center—studies, we have to clearly state that this FLC. 11,33,35,45 Other authors determined the absolute analysis was exploratory. Comparison of studies CSF κ-FLC concentrations only.10,30,35,37,39,45,51,55–57 applying different non-linear formulae11,46,58 did not As the intrathecal κ-FLC fraction is greater than 80% reveal a difference, but the power for this analysis was in most CIS/MS patients,9,46 one might argue that the low due to the small number of studies. So far, there is contribution of blood-derived κ-FLC to the total CSF one study that compared the performance of all three κ-FLC concentration is negligible in cases with formulae within an independent cohort reporting a intrathecal synthesis. In this meta-analysis, we did not diagnostic sensitivity ranging from 96% to 98% in find a statistically significant difference in the diag- MS patients and 40% to 44% in CIS patients.36 nostic performance between both κ-FLC index and IFκ-FLC compared to CSF κ-FLC concentration. At this point, it has to be stated that studies dealt dif- However, the statistical power was below 80% and, ferently with samples in case of non-detectable CSF thus, insufficient to interpret the not-statistically-sig- κ-FLC concentrations. Some studies used the lower nificant results with a small enough Type II error. This detection limit, while others set these samples to means that superiority of κ-FLC index over CSF κ- “zero” or even omitted these samples from the statis- FLC concentration (or even vice versa) cannot be tical analysis. For the absolute CSF κ-FLC concentra- excluded. A recent study further elaborated this tion, samples treated as “zero” or set to the lower research question, separated patients into low and detection limit still means that these samples are in high CSF κ-FLC categories (based on median values) the lower concentration range. Hence, determination and observed that CSF κ-FLC concentration, Qκ-FLC of cut-off values is not affected, and the clinical inter- and κ-FLC index showed similar diagnostic perfor- pretation is clear (i.e. no intrathecal synthesis) as the mance in the high category, but not in the low cate- lower detection limit (e.g. 0.3 mg/L) is by far lower gory with inferiority of CSF κ-FLC and to some than the cut-off (in this meta-analysis 0.96 mg/L). extent also of Qκ-FLC.61 Thus, the impact of serum κ- However, κ-FLC index values depend also on serum FLC and Qalb is indeed negligible in patients with κ-FLC concentration and Qalb, so that different han- high intrathecal κ-FLC synthesis, but probably not in dling of non-detectable CSF κ-FLC concentration patients with only low or modest intrathecal κ-FLC might indeed lead to considerably varying index val- production. This might be of importance in CIS ues, which might then affect cut-off values. patients who showed lower diagnostic sensitivity and lower amount of intrathecal κ-FLC.9 Another very Studies that validate the herein observed cut-offs in a recent large multicenter study including more than multicenter setting are needed. These studies should 1600 patients also reported that κ-FLC index and IFκ- consider different handling in case of non-detectable FLC performed slightly better than absolute CSF κ- CSF κ-FLC values, and the potential impact of differ- FLC concentration.62 Further studies are required to ent assays and platforms as well. We did not find a compare the different κ-FLC measures in patients statistically significant impact of assay and platform with varying degree of intrathecal B cell activity and on κ-FLC index, in line with a recent large multicen- varying blood–CSF-barrier function. tre study that also did not observe any impact of the platform on κ-FLC index.62 Using the ratio of the CSF Different cut-off values for κ-FLC index, for CSF κ- and serum κ-FLC concentration (for calculating the FLC concentration, as well as different formulae index) might be less prone to laboratory variations. defining the Qlim κ-FLC (Presslauer et al.,58 Reiber et The potentially different susceptibility of κ-FLC al.,46 Senel et al.11) for calculating the IFκ-FLC have index and absolute CSF κ-FLC concentrations to 176 journals.sagepub.com/home/msj H Hegen, J Walde et al. laboratory variation should be further addressed. assays; and interpretation of results is rater-depend- Altogether, for calculation of κ-FLC index (or IFκ-FLC) ent.67 It cannot be excluded that OCB would have in case of CSF κ-FLC concentrations below detection shown better performance if tested only in few, spe- limit, we suggest setting the value to the detection cialized laboratories. However, it has to be clearly limit rather than to zero to avoid larger variation stated that one of the clear advantages of κ-FLC is across laboratories. the reliable and rater-independent determination, which should overcome technical difficulties and Diagnostic sensitivities and specificities as reported finally allow a widespread use. by different studies showed a certain variability not only for κ-FLC measures, but also for OCB. This In conclusion, it seems reasonable to consider intrath- arises from a certain heterogeneity of included ecal κ-FLC synthesis equally to CSF-restricted OCB, patients between studies. It is evident that sensitivity both reaching a diagnostic sensitivity and specificity differs whether CIS patients or MS patients are of approximately 90% without significant differences included.64 Specificity is lowered when patients with when meta-analyzed. Statistically sufficient power inflammatory neurological disease (IND) were for the comparisons exists only for κ-FLC index. The included into the control group. κ-FLC in the CSF potential of κ-FLC in the CSF as new biomarker in are—similar to CSF-restricted OCB—a sign of MS was clearly demonstrated. Due to considerable intrathecal inflammation and thus can support the methodological advantages as a fast, time- and labor- diagnosis of MS, but they are not specific for MS. The saving, rater-independent and reliable method, spectrum of diseases which show an intrathecal κ- intrathecal κ-FLC synthesis might serve as alternative FLC synthesis is probably similar to that with CSF- tool to measure intrathecal immunoglobulin synthe- restricted OCB. κ-FLC synthesis reflects IgG sis. A detailed review of the advantages and limita- synthesis, but might be present also in case of intrath- tions of κ-FLC and OCB, respectively, and consensus ecal IgA or IgM synthesis. Studies on the frequency recommendations for implementation of κ-FLC in of intrathecal κ-FLC synthesis in neurological dis- clinical routine are given elsewhere.68 In future, κ- eases other than MS are still rare. Apart from a mix- FLC might be used as a screening test and in certain ture of different IND as part of control populations, constellations OCB as a confirmation test, for exam- dedicated disease-specific studies exists only for a ple, in case of borderline κ-FLC results, as already few entities, for example, neuroborreliosis.65,66 For implemented by some clinical laboratories.55 Since this meta-analysis, we applied a model considering the best algorithm to determine intrathecal κ-FLC not only between-study variation, but also within- synthesis has to be established and universal cut-off study variation and used only studies using both κ- values for different platforms remain to be confirmed, FLC measures and OCB. Therefore, potential sources the combination of both tests—intrathecal κ-FLC and of bias were reduced and allowed a reliable compari- OCB—might be the best option at this moment. son of the above-mentioned parameters. Furthermore, robustness of findings was checked by subgroup anal- Authors’ Contributions yses (different patient groups (CIS, MS, mixed H.H. has contributed in conception and design of the cohorts), different control groups (non-inflammatory study, acquisition of data, analysis and interpretation and inflammatory/non-inflammatory), and different of data, and drafting the manuscript. J.W. has contrib- assays (Freelite, N Latex) and platforms (nephelome- uted in analysis and interpretation of data and revision try, turbidimetry)). of the manuscript for intellectual content. G.A., S.G., B.K., M.K., R.S., C.T., H.T., L.M.V., M.A.V.W., and There are some limitations of the meta-analysis. H.Z. have contributed in revision of the manuscript The statistical power for κ-FLC measures apart for intellectual content. K.B. has contributed in acqui- from the κ-FLC index was low, so that firm conclu- sition of data and revision of the manuscript for intel- sions on the similar diagnostic performance of IFκ- lectual content. F.D. has contributed in conception FLC, Qκ-FLC, CSF κ-FLC concentration, and OCB and design of the study, acquisition of data, analysis cannot yet be drawn. Most of the studies did not and interpretation of data, and revision of the manu- report how their cut-off values were obtained. This script for intellectual content. might have an impact on our estimated cut-off val- ues for κ-FLC index and CSF κ-FLC concentration Declaration of Conflicting Interests (as discussed above). Another limitation is that the The author(s) declared the following potential con- analytic performance of OCB detection probably flicts of interest with respect to the research, author- differed between studies, as different methods were ship, and/or publication of this article: HH has used, for example, commercial versus in-house participated in meetings sponsored by, received journals.sagepub.com/home/msj 177 Multiple Sclerosis Journal 29(2) speaker honoraria or travel funding from Bayer, Siemens and Teva. LMV has served at scientific advi- Biogen, Celgene, Merck, Novartis, Sanofi-Genzyme, sory boards, participated in meetings sponsored by, Siemens, Teva, and received honoraria for acting as received speaking honoraria or travel funding or consultant for Biogen, Celgene, Novartis and Teva. research grants from Roche, Sanofi, Merck, Biogen, GA has received speaking honoraria and compensa- Bristol Myers, and Novartis. MAVW has received tion for consulting services or participation in advi- research grants from The Binding Site, Siemens sory boards from Sanofi, Merck, Roche and Horizon Healthineers and Sebia Inc, has participated in an Therapeutics; travel funding from Novartis, Roche advisory board for Myeloma360. HZ has served in and ECTRIMS; is the editor for Europe of Multiple scientific advisory boards and/or as a consultant for Sclerosis Journal—Experimental, Translational and Abbvie, Alector, Annexon, Artery Therapeutics, Clinical; and is a member of the International Women AZTherapies, CogRx, Denali, Eisai, Nervgen, Novo in Multiple Sclerosis (iWiMS) network executive Nordisk, Pinteon Therapeutics, Red Abbey Labs, committee. KB has participated in meetings spon- Passage Bio, Roche, Samumed, Siemens sored by, received speaking honoraria or travel fund- Healthineers, Triplet Therapeutics, and Wave; has ing from Roche, Biogen, Sanofi, and Teva. SG has given lectures in symposia sponsored by received speaker honoraria and has been a member of Cellectricon, Fujirebio, Alzecure, Biogen, and scientific boards for Biogen Idec, Genzyme, Novartis, Roche; and is a co-founder of Brain Biomarker and Merck and received grant funding from Genzyme, Solutions in Gothenburg AB (BBS), which is a part Merck and Takeda. MK has received funding for of the GU Ventures Incubator Program (outside sub- travel and speaker honoraria from Bayer, Novartis, mitted work). FD has participated in meetings spon- Merck, Biogen Idec and Teva Pharmaceutical sored by or received honoraria for acting as an Industries Ltd. and serves on scientific advisory advisor/speaker for Alexion, Almirall, Biogen, boards for Biogen Idec, Merck Serono, Roche, Celgene, Genzyme-Sanofi, Merck, Novartis Novartis and Gilead. CT has a collaboration contract Pharma, Roche, and Teva. His institution has with ADx Neurosciences, Quanterix and Eli Lilly, received research grants from Biogen and Genzyme performed contract research or received grants from Sanofi. He is section editor of the MSARD Journal AC-Immune, Axon Neurosciences, Bioconnect, (Multiple Sclerosis and Related Disorders). JW, Biogen, Biorchestra, Brainstorm Therapeutics, BK, and RS have nothing to disclose. Celgene, EIP Pharma, Eisai, Novo Nordisk, PeopleBio, Roche, Toyama, and Vivoryon. She serves Funding on editorial boards of Medidact Neurologie/Springer, The author(s) received no financial support for the Alzheimer Research and Therapy, Neurology: research, authorship, and/or publication of this Neuroimmunology & Neuroinflammation, and is edi- article. tor of a Neuromethods book Springer. Research of CET is supported by the European Commission ORCID iDs (Marie Curie International Training Network, grant Janette Walde https://orcid.org/0000-0002-1241- agreement no. 860197 (MIRIADE), Innovative 5292 Medicines Initiatives 3TR (Horizon 2020, grant no. Klaus Berek https://orcid.org/0000-0003-2755- 831434) and JPND (bPRIDE), National MS Society 2043 (Progressive MS alliance) and Health Holland, the Georgina Arrambide https://orcid.org/0000-0002- Dutch Research Council (ZonMW), Alzheimer Drug 2657-5510 Discovery Foundation, The Selfridges Group Sharmilee Gnanapavan https://orcid.org/0000- Foundation, Alzheimer Netherlands, Alzheimer 0003-2817-9922 Association. CT is recipient of ABOARD, which is a Michael Khalil https://orcid.org/0000-0002-5350- public–private partnership receiving funding from 3328 ZonMW (#73305095007) and Health~Holland, Charlotte Teunissen https://orcid.org/0000-0002- Topsector Life Sciences & Health (PPP-allowance; 4061-0837 #LSHM20106). 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