1134217 research-article20222022 MSJ0010.1177/13524585221134217Multiple Sclerosis JournalH Hegen, G Arrambide MULTIPLE SCLEROSIS MSJ JOURNAL Personal Viewpoint Cerebrospinal fluid kappa free light chains for Multiple Sclerosis Journal 2023, Vol. 29(2) 182­–195 the diagnosis of multiple sclerosis: A consensus DOI: 10.1177/ https://doi.org/10.1177/13524585221134217 13524585221134217 https://doi.org/10.1177/13524585221134217 statement © The Author(s), 2022. Article reuse guidelines: sagepub.com/journals- Harald Hegen, Georgina Arrambide , Sharmilee Gnanapavan , Batia Kaplan, permissions Michael Khalil , Ruba Saadeh, Charlotte Teunissen , Hayrettin Tumani, Luisa Maria Villar, Maria Alice V Willrich, Henrik Zetterberg and Florian Deisenhammer Abstract: Cerebrospinal fluid (CSF) analysis is of utmost importance for diagnosis and differential diag- Correspondence to: H Hegen nosis of patients with suspected multiple sclerosis (MS). Evidence of intrathecal immunoglobulin G (IgG) Department of Neurology, Medical University of synthesis proves the inflammatory nature of the disease, increases diagnostic certainty and substitutes for Innsbruck, Anichstraße 35, dissemination in time according to current diagnostic criteria. The gold standard to determine intrathe- A-6020 Innsbruck, Austria. harald.hegen@i-med.ac.at cal IgG synthesis is the detection of CSF-restricted oligoclonal bands (OCBs). However, advances in Harald Hegen laboratory methods brought up κ-free light chains (FLCs) as a new biomarker, which are produced in Florian Deisenhammer Department of Neurology, excess over intact immunoglobulins and accumulate in CSF in the case of central nervous system-derived Medical University of inflammation. Overwhelming evidence showed a high diagnostic accuracy of intrathecal κ-FLC synthesis Innsbruck, Innsbruck, Austria in MS with sensitivity and specificity of approximately 90% similar to OCB. κ-FLCs have advantages Georgina Arrambide Servei de Neurologia- as its detection is fast, easy, cost-effective, reliable, rater-independent and returning quantitative results Neuroimmunologia, Centre d’Esclerosi Múltiple de which might also improve the value of predicting MS disease activity. An international panel of experts Catalunya (Cemcat), in MS and CSF diagnostics developed a consensus of all participants. Six recommendations are given Vall d’Hebron Hospital Universitari, Universitat for establishing standard CSF evaluation in patients suspected of having MS. The panel recommended to Autònoma de Barcelona, include intrathecal κ-FLC synthesis in the next revision of MS diagnostic criteria as an additional tool to Barcelona, Spain Sharmilee Gnanapavan measure intrathecal immunoglobulin synthesis. 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, disease activity, prediction, biomarker, index, consensus 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, Introduction produce light chains in excess of 10%–40% over Graz, Austria Diagnosis of multiple sclerosis (MS) requires the heavy chains and secrete them as free forms (Figure Ruba Saadeh Department of Laboratory combination of clinical signs and symptoms with 1).5,6 Similar to immunoglobulins, free light chains Medicine and Pathology, para-clinical findings obtained by magnetic resonance (FLCs) accumulate in the CSF in the case of chronic Mayo Clinic, Rochester, MN, USA/Department of imaging and cerebrospinal fluid (CSF) analysis.1 inflammatory diseases of the central nervous system Neurology, Mayo Clinic, Evidence of intrathecal immunoglobulin G (IgG) syn- such as MS.7 FLCs were discovered long ago; how- Rochester, MN, USA thesis in the CSF, although not specific for MS,2 sub- ever, their quantitative detection with high sensitivity Charlotte Teunissen Neurochemistry Laboratory, stitutes for dissemination in time according to current and, thus, in low-level compartments such as CSF Department of Clinical diagnostic criteria1 and increases diagnostic certainty was not possible until technological advances at the Chemistry, Amsterdam Neuroscience, Program in the appropriate clinical setting.3 The gold standard beginning of the century.7 The breakthrough was Neuroinflammation, Vrije to prove intrathecal IgG synthesis is the detection of achieved by producing detection antibodies directed Universiteit Amsterdam, Amsterdam UMC, CSF-restricted oligoclonal bands (OCBs).4 against unique FLC epitopes.8 Amsterdam, The Netherlands Hayrettin Tumani B cells produce intact immunoglobulins by assem- A multitude of studies have shown a high diagnostic CSF Laboratory, Department of Neurology, University of bling light chains and heavy chains via disulfide accuracy of the κ-FLC isotype in the CSF to dis- Ulm, Ulm, Germany bonds and non-covalent interactions, but B cells also criminate patients with MS from other neurological 182 journals.sagepub.com/home/msj H Hegen, G Arrambide et al. Luisa Maria Villar Biostatistics Unit, Department of Immunology, Hospital Universitario Ramón y Cajal, Madrid, Spain Maria Alice V Willrich Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Henrik Zetterberg Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden/Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Gothenburg, Sweden/Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK/ UK Dementia Research Institute at UCL, London, UK/Hong Kong Center for Neurodegenerative Diseases, Hong Kong, China Figure 1. Immunoglobulin FLCs as an emerging biomarker for intrathecal B-cell activity. Terminally differentiated B cells produce (A) intact immunoglobulins that consist of bound light chains (green) and heavy chains (blue), as well as (B) in excess FLCs. Both immunoglobulins and FLC serve as a biomarker for B-cell activity. CH: constant heavy chain domain; CL: constant light chain domain: Fab; fragment antigen-binding; Fc: fragment crystallizable; FLC: free light chain; VH: variable heavy chain domain; VL: variable light chain domain. diseases;9 and the detection of κ-FLC has consider- with suspected MS and to provide consensus rec- able methodological advantages compared to the ommendations on the use of κ-FLC in routine diag- detection of OCB.8,10 However, a strong consensus nostic work-up. on the role of κ-FLC as a biomarker in MS is still lacking. This might be due to a certain heterogeneity between published studies ranging from different Methods patient populations, assays, different κ-FLC meas- In October 2021, an international panel convened in ures (e.g. κ-FLC index versus absolute CSF κ-FLC Vienna, Austria, to discuss the use of CSF and, in par- concentration) and cut-off values. ticular, the applicability of κ-FLC in the CSF for rou- tine diagnostic purposes in patients with suspected In an effort to evaluate and recommend the type of MS. The panel was composed of experts in the diag- CSF analysis that yields the greatest diagnostic nosis and management of MS patients and/or CSF sensitivity and specificity for the diagnosis of MS analysis, including neurologists and laboratory medi- considering new technologies in the last two dec- cine specialists from 11 institutions across eight coun- ades, a working group was formed. The aim was to tries and three continents. All participants are listed as produce a report for neurologists and laboratory authors of this manuscript. This meeting was endorsed medicine specialists on what would be considered by the European Committee for Treatment and as ‘standard’ for the evaluation of CSF in patients Research in Multiple Sclerosis (ECTRIMS). journals.sagepub.com/home/msj 183 Multiple Sclerosis Journal 29(2) The panel presented and discussed data from research counts, different WBC subpopulations (e.g. relevant published in English about CSF analysis and, in par- percentages of neutrophils) and higher Qalb and OCB ticular, about κ-FLC in patients with clinically iso- infrequently (only up to 10%–20%).13,14 For details on lated syndrome (CIS) and MS. While specific CSF collection and analysis, we refer to previously recommendations for the implementation of κ-FLC in published consensus guidelines.2,4,15 the diagnostic process for patients with suspected MS were developed during the meeting, the panel set out Consensus statement 1. Neurologists need to con- to create the whole consensus document afterwards. sider the results of all tests performed as part of the Furthermore, the panel decided to perform a system- CSF panel (e.g. white blood cell count, differential atic review and meta-analysis to provide a summary cell profile, albumin quotient, intrathecal Ig synthesis, of the diagnostic value of κ-FLC in patients with CIS CSF/serum glucose ratio or CSF lactate), which and MS and to compare its performance to OCB (pub- should be interpreted in the context of clinical and lished elsewhere Hegen et al.9). imaging findings. The consensus document was first drafted by the prin- cipal author. The first draft was then circulated to all Intrathecal immunoglobulin synthesis panellists, who iteratively contributed to the docu- Different methods are available for the detection of ment until an agreement was reached on the final intrathecal immunoglobulin synthesis in patients document. For each of the recommendations, a mini- with suspected MS, each with certain strengths and mum agreement of 90% (i.e. 11 of 12 co-authors) was limitations. required. If agreement on a recommendation was not achieved, it was discussed again, and a new proposal 1. Quantitative intrathecal IgG synthesis: The re-circulated, until a final agreement was achieved. concentration of total IgG in CSF and serum are determined followed by the calculation of certain formulae such as IgG index,16 Reiber17 Routine CSF panel in MS diagnostic work-up or Auer et al.18 formulae referring patients’ The full spectrum of routine CSF parameters including individual values to a predefined upper normal white blood cell (WBC) count, differential cell profile limit. This approach shows a moderate diag- (assessed, e.g. by inspection of CSF cytology), albu- nostic sensitivity of approximately 70% in MS min quotient (Qalb) and intrathecal Ig synthesis con- patients.11 tributes to the diagnosis of MS and the exclusion of 2. Qualitative intrathecal IgG synthesis: The other causes of CNS inflammation mimicking MS, for detection of oligoclonal IgG bands (OCB) by example, vasculitis, chronic infection or other acquired isoelectric focussing (IEF) followed by demyelinating disorders, such as neuromyelitis optica immuno-detection is currently the gold stand- spectrum disorders (NMOSD) and myelin oligoden- ard method.1,4 This technique compares paired drocyte glycoprotein-associated disorders (MOGAD).4 CSF and serum samples of each individual A recent, comprehensive study on routine CSF param- patient. Intrathecal IgG synthesis is demon- eters in more than 500 patients with CIS and MS strated if OCBs are present in CSF without cor- applying the McDonald criteria 2017 revealed that responding bands in the serum (patterns II and approximately 50% of patients show CSF pleocytosis III) (Figure 2).4 This method shows high diag- with a WBC count up to 40/µL (95th percentile), that nostic sensitivity and specificity both of lymphocytes followed by monocytes are the predomi- approximately 90%,19 however, provides the nant cell type in CSF cytology, that the blood-CSF- qualitative determination of an intrathecal IgG barrier function is abnormal in less than 10% of cases synthesis (i.e. positive or negative).4 with Qalb up to approximately 10 (95th percentile), and 3. Quantitative intrathecal κ-FLC synthesis: κ- that intrathecal IgG synthesis as determined by CSF- FLC concentrations are measured in CSF and restricted OCB are present in up to 95% of patients.11 serum followed by calculation of either the κ- CSF findings slightly differ between the different FLC index or an intrathecal κ-FLC fraction MS disease courses; for example, patients with pro- (IFκ-FLC), where again patients’ individual val- gressive MS show lower WBC counts and less fre- ues are compared to a predefined upper normal quently CSF pleocytosis; and CSF-restricted OCBs limit. An overview of the different κ-FLC are found in approximately 80% of CIS patients but measures investigated in MS is provided in in up to 95% of MS patients10 with lower prevalence Table 1. Certain aspects, for example, consider- in areas of lower geographic latitude.12 Relevant dif- ing only absolute CSF κ-FLC concentrations, ferential diagnoses frequently show higher WBC are addressed below. 184 journals.sagepub.com/home/msj H Hegen, G Arrambide et al. Figure 2. Pattern of oligoclonal IgG bands. Type I No bands in CSF and serum. Type II OCBs present in CSF without corresponding bands in serum. Type III OCBs present in both CSF and serum, with additional bands present in CSF. Type IV OCBs present in CSF, which are identical to those in serum ( ‘mirror pattern’). Type V bands present in CSF, which are identical to those in serum ( ‘ladder pattern’). CSF: cerebrospinal fluid; OCBs: oligoclonal bands; S: serum. Table 1. Various κ-FLC parameters investigated in MS. ■ CSF κ-FLC concentration κ − FLCCSF ■ Qk − FLC = κ − FLC Serum κ − FLCCSF κ − FLC Serum ■ κ − FLC index= Albumin CSF Albumin Serum ■ IF κ-FLC with different underlying formulae to determine the Qalb-dependent upper reference limit (Qlim κ-FLC) and, thus, the limit to define IFκ-FLC - Presslauer et al.:20 Qlim − FLC = 0.9358 × Qalb 0.6687 - Hegen et al.: Qlim − FLC = 3.1276 × Qalb 21 0.8001 - Senel et al.:22 Qlim − FLC = 9.5 + 2.08 × Qalb - Reiber et al.:23 Qlim − FLC = 3.27 × (Qalb 2 + 33)0.5 − 8.2 × 10−3 CSF: cerebrospinal fluid; FLC: free light chain; IF: intrathecal fraction; Qalb: CSF/serum albumin quotient. Diagnostic accuracy of intrathecal κ-FLC and 5800 control subjects. The κ-FLC index showed synthesis in patients with CIS and MS a diagnostic sensitivity ranging from 52% to 100% A systematic review and meta-analysis summarized (weighted average: 88%) and specificity from 69% to the evidence on the diagnostic accuracy of intrathecal 100% (89%). OCB had a diagnostic sensitivity of κ-FLC synthesis to discriminate patients with CIS 37% to 100% (85%) and a specificity of 74% to 100% and MS from other neurological diseases and com- (92%). The comparison of these two parameters by pared its performance to OCB.9 bivariate mixed model – considering between-study and within-study heterogeneity and having a statisti- Most evidence exists for κ-FLC index with 32 studies cal power of 99% – clearly showed that the diagnostic performed on approximately 3300 CIS/MS patients accuracy of κ-FLC index and OCB are similar.9 journals.sagepub.com/home/msj 185 Multiple Sclerosis Journal 29(2) The other parameters previously used to determine and CSF κ-FLC concentration showed similar diag- intrathecal κ-FLC, for example, IFκ-FLC, or the CSF nostic performance in the high category, but not in the κ-FLC concentration, also achieved a diagnostic low category with the inferiority of CSF κ-FLC and to accuracy, which was similar to OCB. However, due to some extent also of Qκ-FLC.62 One might conclude that the low number of studies the statistical power for the impact of serum κ-FLC and Qalb is indeed negligi- these comparisons was smaller than 80% and, thus, ble in patients with high intrathecal insufficient to interpret non-statistically significant κ-FLC synthesis, but probably not in patients with results with a small enough Type II error. For detailed only low or modest intrathecal κ-FLC production. A analyses please refer to Hegen et al.9 very recent large multicenter study including more than 1600 patients also reported that κ-FLC index Consensus statement 2. The single most informative and IFκ-FLC performed slightly better than absolute analysis in MS, although not disease-specific, is the CSF κ-FLC concentration.63 Further studies are assessment of intrathecal immunoglobulin synthesis, required to compare the different κ-FLC measures in either by qualitative detection of a CSF-unique IgG patients with varying degrees of intrathecal B-cell fraction (using IEF followed by immunodetection), or activity, varying blood-CSF-barrier function and var- a quantitative CSF κ-FLC fraction (using nephelom- ying serum κ-FLC concentrations, as the impact of etry or turbidimetry). grossly elevated serum FLC levels or elevated Qalb has not been sufficiently investigated. This might be of interest also in terms of differential diagnosis, as, Different parameters to determine intrathecal for example, NMOSD sometimes shows considerable κ-FLC synthesis blood-CSF-barrier dysfunction,13 which might lead to κ-FLC in the CSF – similar to immunoglobulins higher diffusion of κ-FLC from the blood into CSF or other proteins – originate either from the blood and possibly to false-positive results if only absolute by diffusion across the blood-CSF-barrier, or by CSF κ-FLC concentrations are measured. intra-thecal production under pathological conditions.24 Conceptually, it seems necessary to determine the Consensus statement 3. Methods considering CSF/ locally synthesized κ-FLC fraction separate from the serum κ-FLC concentration and CSF/serum albumin blood-derived fraction as done for total IgG. The quotient, for example, the κ-FLC index, show a good majority of studies used the κ-FLC index22,25–55 or overall agreement with CSF κ-FLC concentrations, IFκ-FLC.22,29,34,38–41,43,48,55–58 Both approaches consider but seem to be superior in cases with low or modest Qalb which is an established marker of the blood-CSF- intrathecal κ-FLC production. barrier function2 and correct for the absolute serum κ-FLC concentration. Few studies used Qκ-FLC.22,37,39,51 Other authors determined the absolute CSF κ-FLC con- Analytic aspects of κ-FLC determination centrations only.33,34,39,41,48,50,51,59–61 As the intra-thecal For the determination of κ-FLC in CSF and serum, originated κ-FLC fraction is greater than 80% in most nephelometry or turbidimetry is widely used and have, CIS/MS patients,23,29 one might argue that the contribu- compared to, for example, enzyme-linked immunoas- tion of blood-derived κ-FLC to the total CSF κ-FLC says, the advantage that automated single measure- concentration is negligible in cases with intrathecal ments are feasible. It was the perception of the panel synthesis. One study reported that around 15% of that nephelometry or turbidimetry is widely accessible. CIS/MS patients showed even higher absolute κ-FLC With regard to the assays, κ-FLC can be measured by concentrations in CSF than in serum that proves an use of either polyclonal (Freelite, The Binding Site, intrathecal synthesis per se.62 Birmingham, UK)8 or monoclonal (N Latex, Siemens, Erlangen, Germany)10 detection antibodies. The above-mentioned meta-analysis compared the performance of κ-FLC index, IFκ-FLC and CSF κ-FLC concentration and observed a similar diagnostic accu- Monoclonal versus polyclonal detection antibody racy. However, the statistical power was below 80% assay on different platforms and, thus, insufficient to interpret non-statistically Polyclonal assays contain anti-κ-FLC antibodies that significant results. Therefore, the superiority of, for are raised in sheep after immunization with a pool of example, κ-FLC index over CSF κ-FLC concentra- human κ-FLC followed by an adsorption step against tion cannot be excluded.9 There is evidence of one intact immunoglobulin (containing bound light recent study that specifically addressed this question, chains), so that only κ-FLC-specific antibodies separated patients into low and high CSF κ-FLC cat- remain in the final antisera.8 In contrast, monoclonal egories and observed that κ-FLC index, IFκ-FLC, Qκ-FLC assays contain a cocktail of multiple monoclonal 186 journals.sagepub.com/home/msj H Hegen, G Arrambide et al. antibodies which are each produced in hybrid cell 90% of patients as determined by the Reiber70 for- lines and finally mixed together.10 Due to these differ- mula, intrathecal κ-FLC synthesis was still not ences in the production, certain characteristics in affected. An explanation is probably the small molec- assay performance arise, for example, a higher lot-to- ular size of κ-FLC of approximately 24 kDa com- lot variation in polyclonal assays.64,65 pared to the larger size of immunoglobulins, for example, of 150 kDa for IgG.71,72 Smaller molecules There are a few studies that compared the polyclonal show a higher CSF/serum ratio, and in the case of arti- (Freelite) and monoclonal (N Latex) assay in serum ficial blood contamination, the relative increase of and found a moderate agreement as determined by CSF concentration is lower. With regard to OCB, correlation analyses (with a coefficient of > 0.9) or blood contamination reduces the chance of detecting Passing–Bablok regression (with a slope between faint bands and might lead to false negative results. 0.74 and 0.99)10,66–68 as well as by concordance rates (sample classification, ranging from 77 up to Free light chains are stable in serum samples frozen at 91%).10,67,68 However, these studies contained sam- −20°C for a storage duration of at least 1 year. ples from patients with, for example, monoclonal Whether thereafter is a relevant change has not been gammopathy covering κ-FLC measurements ranging investigated yet.73 Studies investigating CSF stability up to thousands mg/L.67,68 The differences observed are missing too. between the methods occurred mainly at the upper end of the analytical range.10,67,68 These extremely Patient-related factors might impact κ-FLC concen- elevated concentrations found in the serum of mono- tration. κ-FLC in serum might depend on renal clear- clonal gammopathy patients are not relevant in MS ance, that is, higher serum FLC concentrations were patients without these co-morbidities. found in older patients with decreased renal func- tion.74 Most studies on the biological variation of Scarce evidence on assay comparison exists for serum κ-FLC apart from an underlying disease absolute CSF κ-FLC concentration (Passing–Bablok revealed a small within-subject variation of <10%.75,76 regression, slope of 0.85)66 and κ-FLC index Whether this small within-subject variation can also (Passing–Bablok regression, slope of 0.94) with be extrapolated to CSF levels in MS has to be deter- moderate agreement.68 mined. Recently, it has been shown that high-dose corticosteroids resulted in lower serum FLC concen- The choice of the platform has an impact on serum trations; however, CSF levels and, more importantly, κ-FLC measurement68 and reference intervals69; how- the FLC index were not affected.77 ever, there are no studies addressing the impact on CSF κ-FLC. Cut-off points to determine intrathecal κ-FLC Considering the information provided on the platform synthesis and assay-specific variations, it is plausible to assume Cut-off points might depend on the clinical question, that κ-FLC index might be less prone to laboratory that is, whether an upper reference limit is determined variations compared to absolute CSF κ-FLC concen- in a non-inflammatory control population,78 or if a tration (due to the use of CSF/serum κ-FLC ratio cut-off is determined to discriminate patients with MS resulting in a dimensionless variable). The above- from other inflammatory neurological diseases. Cut- mentioned meta-analysis did not show a statistically off points might also vary depending on whether the significant difference between κ-FLC index across focus is to increase diagnostic sensitivity or diagnos- the methods employing monoclonal or polyclonal tic specificity.66 The impact of laboratory methods on antibodies as reagents in multiple platforms clinically κ-FLC measurements, which again might influence available.9 However, this has to be investigated by cut-off points, has been discussed above. further research. The vast majority of studies using κ-FLC index, CSF κ-FLC concentration, Qκ-FLC as well as different for- Pre-analytics and robustness mulae to define Qlim κ-FLC20,22,23 for calculation of IFκ- κ-FLC in the CSF are by far less susceptible to blood FLC compared CIS/MS patients to heterogeneous contamination, which can occur due to traumatic control populations, and only the minority used pure lumbar puncture, as compared to other CSF proteins, non-inflammatory disease controls. Furthermore, for example, immunoglobulins. A study showed that most of these studies applied a discriminatory cut-off even in case of a blood contamination that led to rather than a cut-off that maximizes either diagnostic false-positive intrathecal IgG synthesis in almost sensitivity or diagnostic specificity.9 journals.sagepub.com/home/msj 187 Multiple Sclerosis Journal 29(2) Table 2. Comparison of CSF-restricted OCB and intrathecal κ-FLC synthesis for diagnosis of MS. κ-FLC synthesis (e.g. κ-FLC index) reflects intrathecal Oligoclonal bands detect intrathecal IgG synthesis IgG synthesis, but also IgA and IgM synthesis PROs PROs ■ High diagnostic sensitivity and specificity ■ High diagnostic sensitivity and specificity ■ High stability in CSF and serum ■ High stability in CSF and serum ■ Robustness ■ Robustness (e.g., blood contamination) ■ Detection of IgG clonality in CSF and serum ■ Easy and fast method compartments (poly-, oligo-, monoclonal) ■ Labour- and cost-effective ■ Differentiation of 2 distinct patterns of intrathecal ■ Quantitative result IgG synthesis ■ Interpretation of results is rater-independent CONTRA CONTRA ■ Time-consuming and technically demanding method ■ Does not differentiate between IgG clonality and ■ Labour-intensive and costly distinct IgG synthesis patterns ■ Qualitative result (i.e. either positive or negative) ■ Interpretation of results is rater-dependent CSF: cerebrospinal fluid; FLC: free light chain; Ig: immunoglobulin; OCB: oligoclonal bands. For the κ-FLC index, reported cut-off values ranged As outlined above, the intrathecal κ-FLC synthesis from 2.4 to 20. In the above-mentioned meta-analysis, a shows a high diagnostic accuracy to discriminate mean cut-off of 6.1 could be determined.9 Even though patients with CIS and MS from other neurological this cut-off is in line with those identified by several diseases with a sensitivity and specificity of approxi- large – partly multicenter – studies,29,33,45,47 it has to be mately 90%, similar to OCB.9 κ-FLC is measured by clearly stated that this was an exploratory analysis. nephelometry or turbidimetry which is – in contrast to the detection of OCB – an easy, reliable, labour-saving, For CSF κ-FLC concentration, a mean discriminatory cost-efficient and rater-independent method.8,10 The cut-off to differentiate CIS/MS patients from controls intrathecal κ-FLC synthesis, for example, by determi- at 0.96 mg/L was observed. Here again, this cut-off is nation of the κ-FLC index, returns a metric result,29 the result of exploratory analysis and is based only on a while OCB status is dichotomous returning either a limited number of studies.9 With regard to the different positive or negative result.4 The quantitative result of non-linear formulae,20,22,23 the small number of studies κ-FLC might gain additional utility in the prediction did not allow any between-study comparisons.9 There of disease activity in early MS (discussed below). is only one study that compared the performance of all three formulae within an independent cohort reporting However, it cannot be differentiated whether similar diagnostic sensitivities ranging from 96% to increased κ-FLC measures are the consequence of 98% in MS patients and 40% to 44% in CIS patients.40 an intrathecal IgA, IgM and/ or IgG synthesis. CSF A comparison in terms of specificity is still lacking. κ-FLC levels do not provide information on the Due to the small number of studies, no cut-off for QFLC clonality of immunoglobulin production and differ- could be determined.9 At this point, we would like to entiation between systemic inflammation with an state that besides laboratory variations, also handling additional intrathecal inflammation (OCB Pattern non-detectable CSF values might have an impact on III), or an isolated intrathecal inflammation (OCB κ-FLC index values. This means that reported cut-off Pattern II) is not possible. This additional informa- values might be biased by this issue. For a detailed dis- tion provided by OCB can be helpful in some clini- cussion, we refer to Hegen et al.9 cal situations, for example, in patients with monoclonal gammopathies and suspected CNS Consensus statement 4. There is extensive data on quite involvement (i.e. Bing Neel syndrome),79,80 or in similar cut-off values for κ-FLC index. However, multi- patients with the CNS involvement of systemic dis- center studies using different platforms and assays should eases (e.g. neurosarcoidosis and systemic lupus ery- be performed to definitively confirm these cut-offs, and thematosus, which show pattern III more frequently certified reference materials should be developed. than MS patients).81 Determination of κ-FLC in the CSF and serum Will κ-FLC replace OCB detection? requires a sample volume of at least 200 µL (due to Determination of intrathecal κ-FLC synthesis and the dead volume in the cuvette placed in the neph- CSF-restricted OCB has certain strengths and limita- elometer or turbidimeter). Even though OCB testing tions (Table 2). requires only a minimum of approximately 10–20 µL 188 journals.sagepub.com/home/msj H Hegen, G Arrambide et al. Figure 3. Strategy reflex algorithm. FLC: free light chain; MS: multiple sclerosis; OCB: oligoclonal bands. (placed on the gel for IEF),19,82 prior determination of cut-offs defining OCB positivity, which is different IgG concentration is recommended so that the appro- numbers of CSF-restricted bands.19 Of interest, there priate dilution of samples can be performed and, thus, are studies reporting that relevant differential diagno- the same amount of IgG applied for the IEF run. ses of MS, for example, NMOSD, show lower κ-FLC levels which might fall into a grey zone (e.g. κ-FLC index of approximately 90 in MS, 20 in NMOSD and Reflex approach 4 in controls).52 Further studies are needed to investi- Determination of intrathecal κ-FLC synthesis might gate patients with low intrathecal κ-FLC levels. be used as a first-line screening test in MS. The reflex approach applies two cut-off points and reports results Studies evaluating the financial aspect of a reflex in case of clearly negative or clearly positive values. approach found the sequential use of κ-FLC as a In the case of values between the two cut-off points screening test and if needed OCB as a confirmation ( ‘grey zone’), OCB detection should follow as a sec- test being less expensive, in terms of reagents, mate- ond step (Figure 3). rial and personnel as compared to OCB detection. Further advantages of the reflex approach still include The low cut-off should ensure that patients with a a reduction in turn-around times and faster reporting negative result have indeed no signs of intrathecal of results.30,34,60 B-cell activity, while the second higher cut-off should unequivocally identify patients with intrathecal B-cell However, as evidence defining the ‘grey zone’ is still activity. While in the majority of CIS and MS patients lacking, one might suggest, at least for the κ-FLC the extent of intrathecal κ-FLC synthesis is high and index, using the lowest and highest cut-off points that the interpretation as a positive result clear, in case of have been published, that is 2.4 and 20 for the κ-FLC low positives, several issues should be considered index, respectively.9 These cut-off points might also including analytic and biological variation. The low be the rationale for further studies. cut-off will label a certain proportion of samples as κ-FLC-positive in the absence of OCB in CSF. Some Consensus statement 5. If results on intrathecal κ- cases will be explained by intrathecal IgA or IgM syn- FLC are borderline, an evaluation by OCB can help to thesis.21 Discrepancies between intrathecal κ-FLC clarify the presence of an intrathecal immunoglobulin synthesis and OCB might also arise from different synthesis, or vice versa. However, until evidence journals.sagepub.com/home/msj 189 Multiple Sclerosis Journal 29(2) Table 3. Six recommendations (commandments) for CSF κ-FLC detection. 1. Neurologists need to consider the results of all tests performed as part of the CSF panel (e.g. white blood cell count, differential cell profile, albumin quotient, intrathecal Ig synthesis, CSF/serum glucose ratio or CSF lactate), which should be interpreted in the context of clinical and imaging findings. 2. The single most informative analysis in MS, although not disease-specific, is the assessment of intrathecal immunoglobulin synthesis, either by qualitative detection of a CSF-unique IgG fraction (using IEF followed by immunodetection), or a quantitative CSF-unique κ-FLC fraction (using nephelometry or turbidimetry). 3. Methods considering CSF/serum κ-FLC concentration and CSF/ serum albumin quotient, for example, the κ-FLC index, show a good overall agreement with CSF κ-FLC concentrations, but seem to be superior in cases with low or modest intrathecal κ-FLC production. 4. There is extensive data on quite similar cut-off values for κ-FLC index. However, multicenter studies using different platforms and assays should be performed to definitively confirm these cut-offs, and certified reference materials should be developed. 5. If results on intrathecal κ-FLC are borderline, an evaluation by OCB can help to clarify the presence of an intrathecal immunoglobulin synthesis, or vice versa. However, until evidence defining borderline intrathecal κ-FLC synthesis is established, the combination of both tests might be the best option at this moment. 6. Determination of intrathecal κ-FLC synthesis should be included in the next revision of MS diagnostic criteria as an additional tool to measure intrathecal immunoglobulin synthesis. CSF: cerebrospinal fluid; FLC: free light chain; IEF: isoelectric focussing; MS: multiple sclerosis; OCB: oligoclonal bands. defining borderline intrathecal κ-FLC synthesis is Author contributions established, the combination of both tests might be H.H. has contributed in conception and design of the the best option at this moment. study, acquisition of data, analysis and interpretation of data and drafting the manuscript. G.A., S.G., B.K., M.K., R.S., C.T., H.T., L.M.V., M.A.V.W., H.Z., and Further research issues F.D. have contributed in conception and design of the Prognostic value of intrathecal κ-FLC synthe- study, acquisition of data, analysis and interpretation sis. There are only a few studies on the predictive of data and revision of the manuscript for intellectual value of κ-FLC index in MS. An overview is given in content. Hegen et al.7 The majority of studies reported that intrathecal κ-FLC synthesis is associated with con- Declaration of Conflicting Interests version from CIS to MS28,35,40,53,83 and that the extent The author(s) declared the following potential con- of intrathecal inflammation as reflected by the κ-FLC flicts of interest with respect to the research, author- index predicted the time to conversion to MS as well ship and/or publication of this article: HH has as disability progression.83,84 Two recent studies participated in meetings sponsored by, received showed in a multivariate approach considering other speaker honoraria or travel funding from Bayer, already known risk factors such as baseline MRI Biogen, Celgene, Merck, Novartis, Sanofi-Genzyme, lesions that in patients with a first CNS demyelinating Siemens, Teva, and received honoraria for acting as event, high κ-FLC index is an independent risk factor consultant for Biogen, Celgene, Novartis and Teva; for early second clinical attack54,85 and fulfilment of GA has received speaking honoraria and compensa- 2017 McDonald criteria.85 These findings fit to previ- tion for consulting services or participation in advi- ous studies that reported a predictive value of IgG sory boards from Sanofi, Merck, Roche and Horizon index86,87 and the number of OCB,87–89 that is, the Therapeutics; travel funding from Novartis, Roche extent of intrathecal inflammation, with future MS and ECTRIMS; is the editor for Europe of Multiple disease activity. In clinical practice, intrathecal κ- Sclerosis Journal – Experimental, Translational and FLC synthesis might serve as a predictive biomarker Clinical; and is a member of the International Women in MS, that is, it could – together with other surrogate in Multiple Sclerosis (iWiMS) network executive markers such as MRI – identify patients in need of committee; SG has received speaker honoraria and early, highly efficacious disease-modifying treatment has been scientific boards from Biogen Idec, and, thus, facilitate treatment decision-making. Genzyme, Novartis and Merck and grant funding from Genzyme, Merck and Takeda; BK has nothing Consensus statement 6. Determination of intrathecal κ- to disclose; MK has received funding for travel and FLC synthesis should be included into the next revision speaker honoraria from Bayer, Novartis, Merck, of MS diagnostic criteria as an additional tool to measure Biogen Idec and Teva Pharmaceutical Industries Ltd. intrathecal immunoglobulin synthesis (Table 3). and serves on scientific advisory boards for Biogen 190 journals.sagepub.com/home/msj H Hegen, G Arrambide et al. Idec, Merck Serono, Roche, Novartis and Gilead; RS Biogen and Genzyme Sanofi. He is section editor of has nothing to disclose; CT has a collaboration con- the MSARD Journal (Multiple Sclerosis and Related tract with ADx Neurosciences, Quanterix and Eli Disorders). Lilly, performed contract research or received grants from AC-Immune, Axon Neurosciences, Bioconnect, Funding Biogen, Bioorchestra, Brainstorm Therapeutics, The author(s) received no financial support for the Celgene, EIP Pharma, Eisai, Novo Nordisk, research, authorship and/or publication of this article. PeopleBio, Roche, Toyama, Vivoryon. She serves on editorial boards of Medidact Neurologie/Springer, ORCID iDs Alzheimer Research and Therapy, Neurology: Georgina Arrambide https://orcid.org/0000-0002- Neuroimmunology and Neuroinflammation, and is 2657-5510 editor of a Neuromethods book Springer. Research Sharmilee Gnanapavan https://orcid.org/0000- of CET is supported by the European Commission 0003-2817-9922 (Marie Curie International Training Network, grant Michael Khalil https://orcid.org/0000-0002-5350- agreement No 860197 (MIRIADE), Innovative 3328 Medicines Initiatives 3TR (Horizon 2020, grant no Charlotte Teunissen https://orcid.org/0000-0002- 831434) and JPND (bPRIDE), National MS Society 4061-0837 (Progressive MS alliance) and Health Holland, the Florian Deisenhammer https://orcid.org/0000- Dutch Research Council (ZonMW), Alzheimer Drug 0003-4541-8841 Discovery Foundation, The Selfridges Group Foundation, Alzheimer Netherlands, Alzheimer Association. CT is a recipient of ABOARD, which is a public-private partnership receiving funding from References ZonMW (#73305095007) and Health–Holland, 1. Thompson AJ, Banwell BL, Barkhof F, et al. Diagnosis Topsector Life Sciences and Health (PPP-allowance; of multiple sclerosis: 2017 revisions of the McDonald #LSHM20106). 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