Assessment of Intrathecal Free Light Chain Synthesis: Comparison of Different Quantitative Methods with the Detection of Oligoclonal Free Light Chains by Isoelectric Focusing and Affinity-Mediated Immunoblotting

Objectives We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. Methods We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. Results Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. Conclusions Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance.


Introduction
with the same ELISA method using purified human myeloma fLC standards (25.7 ± 10.5 mg/L and 4.34 ± 2.0 mg/L for fKLC and fLLC, respectively) [30]. Nevertheless, the values obtained by the same group using the same (monoclonal) standards but different ELISA method with two anti-fLC specific antibodies [31] gave even more different results (3.11 ± 1.18 mg/L and 2.30 ± 1.03 mg/L for fKLC and fLLC, respectively). These discrepancies might be related to the second problem of fLC assays: even minor cross-reactivities of the antibodies with bound light chains could result in significant overestimation of fLC in biological material where the concentration of immunoglobulins is approximately a thousand times larger than that of the fLC [32]. In addition, it has been shown that some antibodies employed in fLC assays detect better fLC dimers rather than monomers, while the degree of fLC dimerisation can vary under pathological conditions [33].
Third, all of the fLC tests have primarily been developed to help in the diagnosis and monitoring of plasma cell dyscrasias, while their performance in other settings has not yet been sufficiently validated.
Finally, fLC concentrations in normal CSF are often so low that they cannot be measured even by the modifications of the original assay. Even with latex particle enhancement, turbidimetry or nephelometry simply come to their limits on principal grounds at concentrations below 0.1 mg/L.
Reports on the diagnostic performance of CSF fKLC measurement in the context of MS diagnosis are extraordinarily consistent. Almost all studies showed performance similar to, or even marginally better than, the detection of oligoclonal IgG bands, irrespective of what the qualitative or quantitative methods were used and whether CSF fKLC concentration, CSF/ Serum quotient, fKLC index (fKLC quotient/Albumin quotient) or any kind of non-linear relationship between fKLC quotient and albumin quotient was used for evaluation. The value of CSF fLLC measurement is less clear. While the earliest reports found intrathecal fLLC synthesis more frequently than that of fKLC [11,12,17], the reverse is observed in almost all later studies, with one notable exception [34]. Some groups were unable to develop a suitable fLLC assay [20,35]. Other groups failed to report their fLLC results although they had apparently performed the CSF fLLC measurement [9] making one guess that the relative scarcity of fLLC data might be caused by some methodical problems and consequently a publication bias.
We therefore aimed at choosing the most suitable method for the routine detection of intrathecal fLC synthesis. After we had succesfully introduced oligoclonal fLC detection [36,37], we looked for a quantitative method for comparison and possibly monitoring purposes. Our sandwich ELISA, which was developed during several years, is based on the same principle as the o-fLC test [38]. After being CE marked for CSF fLC analysis, we started to use the Freelite assay in our laboratory [39] and aimed to compare it with other commercially available or inhouse developed tests. The present paper is intended to report the analysis of such a comparison and to point to some questions that arose during this research.

Patients and samples
Paired CSF and serum samples of 166 patients were included in the analysis. We categorised the patients into diagnosis groups as follows: 1. multiple sclerosis (MS; n = 17), 2. clinically isolated syndrome (CIS; n = 30), 3. other inflammatory neurological diseases (OIND; n = 20), 4. non-inflammatory neurological diseases (NIND; n = 57), 5. no evidence of organic nervous system disease (symptomatic controls according to the definition of Teunissen et al. [40]; n = 18), and 9. unknown or uncertain diagnoses (n = 24). Three CIS patients had asymptomatic gadolinium-enhancing lesions on brain magnetic resonance imaging (MRI) defining definite MS in the latest version of McDonald's diagnostic criteria [41] at the time of lumbar puncture. Nevertheless, the patients were still categorised within the CIS group for the purpose of this study because they experienced the first attack of the disease shortly before the time of lumbar puncture. Other eight CIS patients progressed into definite MS during the period of sample recruitment (May 2014 -August 2015) or data analysis (September-October 2015). The diagnoses in the OIND group comprised meningoencephalitis (n = 3), encephalitis (n = 1), acute disseminated encephalomyelitis (n = 1), myelitis (n = 2), neuroborreliosis (n = 2), neurosyphilis (n = 1), neurolupus (n = 1), neuromyelitis optica spectrum disease (n = 1), plexitis (n = 1) and immune-mediated polyneuropathies (n = 7).
Patients with MS together with those CIS patients that fulfilled the recent definite MS criteria at the time of lumbar puncture or had progressed into definite MS before the diagnosis data were collected and analysed formed the MS group (n = 28) to be compared with the non-MS group consisting of patients of the groups 3, 4 and 5 (n = 95). For the comparison between inflammatory neurological diseases (IND) and non-inflammatory neurological diseases (NIND), we compared the MS group described above plus patients of the group 3 (n = 48) against patients in the groups 4 and 5 (n = 75). CIS patients that did not progress into definite MS during the study period as well as patients with an unknown or uncertain diagnosis were excluded from these analyses.
CSF was drawn by lumbar puncture and venous blood by venipuncture under standard conditions. Within one week after CSF withdrawal, immediately after all routine tests including oligoclonal IgG detection had been performed, paired CSF and serum samples were frozen to -30˚C and thawed only once just before the fLC quantitative analysis. CSF storage at +2 to +8˚C for up to 7 days is allowed in the package insert of the Freelite SPA PLUS kits (method A, see below). Additionally, we have tested 11 CSF samples frozen either immediately or after 7 days at +2˚C to +8˚C and found only marginal decrease of fKLC concentrations and no decrease of fLLC concentrations (detailed data are available from Dryad, doi: 10.5061/dryad. 8tk26). For oligoclonal fLC detection, an aliquot was kept at +2 to +8˚C for up to 20 days. We have previously reported that oligoclonal fLC patterns remain stable during this period [37].
All the patients signed the informed consent for anonymised use of the surplus of the fluids for research purposes. The study was approved by the Ethics Committee of the University Hospital Ostrava (Ref. No. 319/2014).

Methods
Free light chain quantitative assays. (A) Turbidimetric assay on SPA PLUS with Freelite Kappa SPA PLUS kit and Freelite Lambda SPA PLUS kit (Cat. No. LK016.L.S and LK018.L.S, respectively, The Binding Site Ltd., Birmingham, UK). These tests use polyclonal sheep antibodies coated onto polystyrene latex. Calibrators are serum-based. The assay was performed according to the manufacturer's instructions. CSF is analysed neat and if the concentration measured exceeds that of the highest standard, the analysis is repeated after manual dilution 1/ 10. The assay uses an extrapolated calibration curve with triplicate measurement of blank and standard 1 (lot-specific, about 0.35 mg/L for fKLC and 0.45 mg/L for fLLC), enabling measurement down to 0.1 mg/L. Values lower than 0.1 mg/L were analysed as 0.09 mg/L in this study. The highest calibrator has concentration about 16 mg/L (lot-specific) for both fKLC and fLLC.
(B) Nephelometric assay on BN ProSpec with N Latex FLC kit (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany, catalogue numbers OPJA03 -OPJF03). The assay uses a mixture of mouse monoclonal antibodies covalently coupled to polystyrene particles. Standards contain purified polyclonal fLC in PBS with 1% human serum albumin [28]. Based on our preliminary experiments, the lowest calibration point was deleted for CSF fKLC analysis on our request by the Siemens technician in order to avoid too many repetitive dilutions. Then the calibration curve spanned the range between 0.0616 and 1.97 mg/L for CSF fKLC, 0.0308 and 0.985 mg/L for Serum fKLC and 0.104 and 3.32 mg/L for both CSF and Serum fLLC. CSF is analysed neat and as soon as the signal exceeds that of the highest standard, automatic dilutions 1/5, 1/20 and 1/100 are performed by the instrument until the measured signal falls within that of the standards. Serum is diluted 1/100 for fKLC and 1/20 for fLLC analysis.
(C) Human Immunoglobulin Free Light Chains Kappa and Lambda ELISA kit (BioVendor-Laboratorni medicina a.s., Brno, Czech Republic, Cat. No. RD194088100R). The wells are pre-coated with monoclonal anti-human immunoglobulin fKLC or fLLC antibodies. The standards are human serum based. CSFs were diluted 1/2 (or more, if appropriate) and sera 1/200. The test was performed according to manufacturer´s instructions. Both standards and samples were analysed in duplicate. The manufacturer provides no information concerning the source of antibodies and it is not clear whether the second antibody is anti-free light chain specific or whether it detects free as well as bound light chains. Six calibrators prepared by subsequent two-fold dilutions span the concentration range 10-320 μg/L for fKLC and 17.5-560 μg/L for fLLC.
(D) In-house ELISA. Microtiter plates (F96MaxiSorp F96, Nunc A/S, Roskilde, Denmark, Cat. No. 442404) were coated either with 0.75 mg/L anti-human free kappa light chain antibody (DAKO, Glostrup, Denmark, Cat. No. A0100) or 0.30 mg/L anti-human free lambda light chain antibody (DAKO, Cat. No. A0101) diluted in carbonate-bicarbonate buffer, pH 9.5 ± 0.1. The plates were covered with paraffin foil and incubated for 18-48 hours in 4˚C. Next, plates were washed twice with phosphate-buffered saline (PBS; pH 7.3±0.1)-0.05% Tween 20 (PBST) and blocked with 1% (w/v) bovine serum albumin (BSA, fraction V receptor grade; Serva Electrophoresis GmbH, Heidelberg, Germany, Cat. No. 11924) in PBST for 75 minutes. CSF and serum were diluted with 0.2% (w/v) BSA in PBST. Usual dilution was 1/10 for CSF and 1/1000 for serum. Samples with high or low fLC content were diluted as appropriate in order for the signal to fall within the calibration curve. For calibration, monoclonal free light chains (Bethyl Laboratories, Montgomery, Texas, USA, Cat. No. P080-126 and P080-127) were used. Seven two-fold dilutions (concentration range between 1.25 and 80.00 μg/L for fKLC and 2.5 and 160.0 μg/L for fLLC) were prepared. The plate was washed 5 times with PBST. Standards and samples were applied in duplicate and the plate was incubated for 2 hours in room temperature. Next, the plate was washed 5 times with PBST, while the biotinylated anti-fLC antibody prepared as described earlier [36] diluted 1/10,000 (0.4 mg/L) in 0.2% BSA. After 90-minute incubation, decantation and washing, horseradish peroxidase streptavidin (ELISA grade, Vector Laboratories, Burlingame, CA, USA, Cat. No. SA-5014) diluted 1/ 750 was applied and the plate was incubated for 40 minutes and washed as described above. Finally, TMB one-step substrate system (DAKO, Cat. No. S159985) was applied and the plate was incubated until suitable colour intensity developed (18-20 minutes). After stopping the reaction with 50 μl of 1M H 2 SO 4 , the optical density was read by DSX ELISA automatic analyser (Dynex, Buštěhrad, Czech Republic). The 4-parameter calibration curve was constructed using the analyser software.
(E) In-house ELISA as described in (D) but using appropriately diluted (1/200) Freelite standards 1-6. The 4-parameter calibration curve was constructed using the in-house developed programme. Calibration range was about 1.8 and 80 μg/L for fKLC and 2.25 and 80 μg/L for fLLC with slight lot-to-lot variations.
Our sandwich ELISA was developed during several years [38] and uses a principle that is similar to the method of qualitative detection of oligoclonal fLC bands by means of affinitymediated immunoblotting, described by Sindic and Laterre [16] and later modified by us [36,37]. Both intra-and interassay coefficients of variations (CVs) were determined for Freelite control samples. In addition, interassay CVs were determined in several pooled CSF and serum samples. Recovery was determined by spiking CSF and serum samples with 10 vol.% of either Bethyl monoclonal light chains or Freelite serum-based standard. Details concerning analytical characteristics of the in-house methods are available from Dryad, doi: 10.5061/dryad.8tk26 Detection of oligoclonal fLC (reference method). Isoelectric focusing (IEF) in 1.2% agarose gels containing 12% sorbitol (interelectrode distance 8.5 cm, limits 200 V/cm, 100 mA, 10 W) was carried out on Multiphor II apparatus (GE Healthcare UK Ltd., Buckinghamshire, England) at 10˚C for 1,200 Vh. 7.5 μl of the respective monoclonal fLC protein (Bethyl, Cat. No. P080-126 for fKLC and P080-127 for fLLC) diluted to 2.0 and 0.2 mg/L were run as positive controls, while intravenous immunoglobulin preparation (Flebogamma, Instituto Grifols S.A., Barcelona, Spain, or Kiovig, Baxter AG, Wien, Austria) diluted to 250 or 500 mg/L IgG was run as a negative control. CSF samples were used neat and serum samples were diluted 1/ 100. All dilutions were performed with 0.75% saline. Paired CSF and serum samples were applied side by side using applicator strips (Serva, Cat. No. 42899). After IEF, fLC were blotted onto nitrocellulose membranes (Amersham™ Hybond ECL, Cat. No. RPN303D, or Amer-sham™ Protran™ Premium, Cat. No. 10600003, GE Healthcare, pore size 0.45 μm) precoated with anti-fLC antibodies (DAKO, Cat. No. A0100 for fKLC and A0101 for fLLC) and blocked with 3% BSA in tris-buffered saline (75 mM TBS, pH 7.5 ± 0.1) for 60 minutes. Next, membranes were washed briefly in phosphate-buffered saline, followed by the fixation of proteins on the membrane with 0.25% glutardialdehyde for 15 minutes at 4˚C. Then, the membranes were briefly rinsed in deionised H 2 O, washed 3 times in TBS, re-blocked with 0.3% BSA in TBS for 15-20 minutes, washed once with TBS and incubated with biotin-labelled anti-fLC antibody (diluted to 5 mg/L) for 105 ± 5 minutes. After washing once in TBS, 3 times in TBST and 2 times in TBS, incubation followed with Alkaline phosphatase streptavidin (Vector, USA, Cat. No. SA-5100) diluted 1/750 for 50 minutes. Next, the membranes were washed twice in TBS, 3 times in TBST and 2 times in TBS. Finally, colour reaction was developed using Alkaline Phosphatase Substrate IV Kit-BCIP/NBT (Vector, USA, Cat. No. SK-5400) for 30 minutes. Membranes were then washed in TBS for 5 minutes and in deionised water for 1-2 minutes. After being held for 1 minute in a vertical position to let the water drop out, the membranes were dried overnight between 2 sheets of filter paper covered with a glass plate.

Statistical analysis
Statistical analysis was performed using MedCalc statistical software version 14.12.0 (MedCalc software bvba, Ostend, Belgium; http://www.medcalc.org; 2014). Kolmogorov-Smirnov test was used for the assessment of normality whenever appropriate. Differences between the methods were assessed by means of Spearman´s correlation coefficient, log-log scatter diagrams, Passing and Bablok regression and Bland-Altman plots. ROC curves were analysed and compared, if appropriate, for the prediction of a positive o-fLC test as well as the MS or IND diagnosis. Correlations between categorical variables (o-IgG and o-fLC) were investigated by the Chi-square test.

Oligoclonal IgG and oligoclonal fLC results
The results of these tests in patients within individual diagnosis groups, MS/non-MS and IND/ NIND groups are provided in S1 Table.

Quantitative method comparison
The comparison of CSF/Serum quotients determined by various methods is presented in Fig  1. For in-depth comparison of CSF and serum concentrations as well as CSF/Serum quotients see Tables A-C in S1 File, Figures A-F in S2 File and Figures A-F in S3 File. Both systematic and proportional differences occurred. The largest differences were observed between the results of the commercially available ELISA method in the CSF that were much lower than those of all other tests. Nevertheless, the obtained concentrations were comparable with those published previously [9]. Serum fLLC were measured much higher by nephelometry using N Latex FLC kit than by turbidimetry using Freelite assay, although there was no significant difference between the two methods when measuring fLLC in CSF.
To get some insight into the possible reasons of differences among the methods used, we analysed standards used in the individual assays by the in-house ELISA method. Optical densities of the standards diluted to similar fLC concentrations differed substantially, especially in the case of fKLC standards. For more details see the S2  ELISAs employed with AUCs 0.87 in all these four cases. There was no significant difference in the AUCs between CSF concentrations, CSF/Serum quotients or indices. When the cut-off for CSF-restricted o-fKLC bands was raised to !6 bands as in our previous studies [37,39], all AUCs for CSF fKLC, fKLC quotient as well as fKLC index were >0.95. For the Freelite method on SPA PLUS , calculated cut-off values were >0.78 mg/L for CSF fKLC, >43.19 Á 10 3 for fKLC quotient and >6.07 for fKLC index, i. e. similar to those found in our preliminary study [39]. However, we feel that while searching for optimal cut-offs may be important for diagnostic purposes, the canonical definition of !2 CSF-restricted oligoclonal bands is rather appropriate for a methodical study. The optimal cut-off values calculated by the ROC curve analysis differed substantially among the methods (Table 1).
Further, the ROC curves for the Freelite™ method on SPA PLUS were compared pairwise with the ROC curves for all the other methods. The AUCs were significantly larger for the Freelite™ method on SPA PLUS than for in-house ELISA using either monoclonal or polyclonal fLC standard only in the case of fLLC CSF concentration and quotient.

ROC curves for the prediction of MS or inflammatory neurological disease diagnoses
Concerning the diagnostic performance of quantitative fLC data in the context of MS or inflammatory neurological disease (IND) diagnoses, fKLC AUCs were significantly larger than those of fLLC. Especially in the context of MS diagnosis, the use of fLC indices was shown to be superior to CSF concentrations or even CSF/Serum quotients. No significant difference between the diagnostic performance of the best quantitative fKLC measures, o-fKLC and o-IgG was revealed (Fig 2). Using the classical criterion of !2 CSF-restricted oligoclonal bands, sensitivities and specificities for MS were 85.   fKLC/fLLC ratios in CSF and sera

Free lambda light chains n (o-fLLC+/ o-fLLC-) CSF fLLC (mg/l) fLLC quotient (Á10 3 ) fLLC index
In the absence of intrathecal fLC synthesis, CSF fKLC/fLLC ratios were slightly lower than the corresponding serum ratios. This result was consistent in all 5 used methods. Thus CSF/Serum quotient of these ratios [(CSF fKLC/fLLC)/(Serum fKLC/fLLC)] was slightly below 1 and the value of 1.0 corresponded approximately to the 75th percentile (Fig 3). About half of MS and CIS patients have a profoundly increased CSF fKLC/fLLC ratio normalised to serum fKLC/ fLLC ratio as discussed above (Fig 4).

Can CSF/Serum fLC quotients be regarded as method-independent values?
According to Reiber [42], CSF/Serum immunoglobulin quotients are method-independent values, provided that the concentrations in both fluids are measured within the same run on the same calibration curve. Nevertheless, despite good overall correlation of the data, we observed considerable differences between fLC quotient values, implying that different and method-specific cut-offs have to be used for the assessment of intrathecal synthesis. We conclude that given current analytical possibilities, quotient method-independence does not apply for fLC.

Relationship between Q fLC and Q Albumin
We confirmed our earlier observations [39] that for both fKLC and fLLC there is a clear correlation between fLC quotient and albumin quotient values in cases with a negative o-fLC test.
Conversely, no such a correlation was observed in cases with CSF-restricted o-fLC bands. This demonstrates that compared to the intrathecal fraction, the relative amount of serum-derived fLC fraction in the CSF becomes negligible, which is in line with the recent study of Presslauer et al. [8] where fKLC intrathecal fraction was close to 100% in most MS patients. This probably accounts for the fact that simple CSF fLC concentration has similar diagnostic sensitivity and specificity as quotients and indices, although we still recommend the use of the latter because false positive results of CSF fLC may occur in cases of very high serum fLC concentrations (e.g. in patients with paraproteinaemia or systemic inflammation). The relationship between fLC quotient and albumin quotient appears to be non-linear and the intrathecal synthesis could be calculated using either inverted Reiber's hyperbole, as we proposed recently [39], or the power function proposed by Presslauer et al. [7,8]. We demonstrated that almost all values lie above the line of equality, as expected for the CSF/Serum quotient of a smaller molecule plotted on the ordinate and that of a larger molecule plotted on the abscissa [43]. The shape of the curve displaying the non-linear relationship is also consistent with the theoretical expectations. However, more patients with negative o-fLC results, especially those with severe blood-CSF barrier disturbance, should be examined so that reliable parameters of these equations could be calculated.

fKLC/fLLC ratios in CSF and sera
Besides CSF/Serum quotient, also fKLC/fLLC ratio can be calculated from the data. Since this ratio is used in serum as an aid in diagnosing and monitoring patients with monoclonal gammopathies, it might be interesting to detect a skewed distribution of fLC in CSF. Our results are in accordance with older studies that showed substantially higher CSF fLLC than fKLC concentrations in the control groups [19,21] but theoretically unexpected. fKLC are assumed to circulate predominantly as monomers, while fLLC as dimers. Hence, fKLC monomers with a lower molecular weight (22.5 kDa) should diffuse into the CSF more easily than fLLC dimers (45 kDa) and, consequently, the values above 1.0 should be obtained. The discrepancy between expected and real data suggests that either there is a general methodological problem in fLC analyses or that there might be a more complex monomer/dimer/oligomer fLC balance than it is currently percieved. Another explanation includes the presence of other physiological factors hindering fKLC or facilitating fLLC diffusion from blood into the CSF. Monomer/dimer patterns of fLC were studied in serum as well as in the CSF [33,44,45] and although these tests seem to be too complicated to be introduced into clinical routine, in further research studies they should complement quantitative determinations of fLC in order to solve this interesting question.
Since the intrathecal fLLC synthesis is usually accompanied by the fKLC synthesis, it may seem logical to drop fLLC studies and perform only fKLC tests. However, from the immunological point of view, the analysis of both fLC might be useful as widely different fLLC values were seen in MS patients by our group [39] as well as by the others [3,4]. It is plausible to suggest that the fKLC/fLLC ratio may provide important additional information on the intrathecal humoral immune response and might even be of prognostic significance. The skewed CSF fKLC/fLLC ratio may be the result of receptor editing/receptor revision [46,47] that has already been described as occurring within the CNS of MS patients [48].

Choosing the method: Practical considerations
Each method as well as each analyser has its own limits and advantages. Finally, we chose the Freelite turbidimetric assay on SPA PLUS analyser, which is economic and user-friendly; manual dilution of the CSF sample is rarely required for fKLC and almost never for fLLC. Nevertheless, ELISA based assays might be better applicable in some laboratories worldwide where access to automated instruments is limited. In addition, diluted CSF is used in our in-house ELISA assay, which leads to a much lower amount of sample required for the analysis. Although the sample volume in the automatised assays is low (SPA PLUS requires 15 μl for fKLC and 6.5 μl for fLLC analysis, i.e. 21.5 μl for both measurements, while 50 μl-i.e. 100 μl for both measurements-were required for the analysis on BN ProSpec), we have to add the dead volume that is slightly above 100 μl in both cases. Nephelometric method on BN ProSpec (with either Freelite or N Latex FLC) might be best suited for scientific purposes. Besides having marginally better sensitivity for fKLC (about 0.05 mg/L versus 0.1 mg/L on the SPA PLUS instrument), in consequent dilutions it strives to achieve a signal in the sample that is close to the middle of the calibration curve where the measurement can be expected to be most precise. On the other hand, this results in a high consumption of reagents for samples with high CSF fLC concentrations, which is a major drawback of this method.

Limits of the study
We are aware of several weaknesses of our study. First, it was impossible to perform all tests on every sample, mainly due to insufficient amounts of CSFs and/or sera needed for the assays. Testing by the N Latex FLC method on BN ProSpec had to be interrupted prematurely because of quality control failure regarding a new lot of reagents that was unfortunately only discovered after thawing a proportion of samples. Simultaneously performing in-house ELISA methods with monoclonal and polyclonal (Freelite™) standards, we found out there were rare cases when one sample showed a signal higher than that of the highest standard in one method, but within the calibration range in the other method. However, the analysis could not be repeated in higher dilution either due to insufficient amount of sample or for logistic reasons.
Second, although theoretically sound, the assumption that the detection of CSF-restricted oligoclonal immunoglobulin or fLC bands represents a gold standard to prove intrathecal synthesis, against which quantitative measures should be compared, was only sufficiently validated for IgG. The negative result of oligoclonal IgG detection is often used as a reference method for detecting the absence of intrathecal humoral immune reaction, but it was shown that o-fLC could sometimes be detected in the absence of o-IgG, especially in other inflammatory CNS diseases rather than MS [16,37]. Therefore, we tried to validate CSF fLC quantitation against o-fLC detection as the reference method. However, unlike for IgG, the amounts of fLC in paired CSF and sera are not matched in the o-fLC assays published so far (including that of ours). Instead, neat CSF and arbitrary diluted serum (1/100) are used. This approach seems more appropriate than the dilution of serum and CSF to the same IgG concentration used in several older studies [11,12,16] because fLC have a smaller molecular weight and therefore a considerably larger serum fraction passes into the CSF. In patients with severe blood-CSF-barrier disturbance, however, systemic o-fLC response might still be falsely detected as intrathecal if serum fLC concentrations are not elevated. In our experience, however, systemic o-fLC response is detected much less frequently than systemic o-IgG response.

Conclusions
In spite of considerable proportional as well as systematic differences between various quantitative fLC assays compared in this study, no major differences were observed regarding the prediction of a positive o-fLC test or the diagnostic performance in the context of inflammatory nervous system disease diagnosis. The performance of o-fKLC as well as quantitative fKLC tests is comparable to that of o-IgG, but not superior. In the context of MS diagnosis, o-IgG seems to be more specific than fKLC tests. We therefore conclude that it is too early to recommend abandoning the o-IgG test as suggested in some recent reports [3,8] until more data have been gathered from various laboratories and different fKLC assay methods have been used. The diagnostic performance of qualitative as well as quantitative fLLC tests is inferior to that of o-IgG and fKLC tests, but enormous variability in the CSF fLLC concentration observed among individual MS patients warrants further studies including prospective evaluation of the possible significance of intrathecal fLLC synthesis in MS. Besides, the fKLC/fLLC ratio in normal CSFs seems to be lower than theoretically expected, based on the postulated predominance of circulating fKLC monomers and fLLC dimers and on the Reiber's blood-CSF barrier diffusion theory [49], which remains to be elucidated.  Table. Cut-offs in the context of multiple sclerosis diagnosis. a. Free kappa light chains b. Free lambda light chains MS, multiple sclerosis; non-MS, other diagnoses than multiple sclerosis or clinically isolated syndrome It should be noted that the number of samples examined by the N Latex FLC™ and BioVendor ELISA is too small to permit definitive conclusions regarding the performance of these tests in the context of MS diagnosis. Ã The second best cutoff value in case the calculated cut-off resulted in 50% sensitivity or specificity that was considered as unacceptable. (RTF) S3 Table. Cut-offs in the context of inflammatory neurological disease diagnosis. a. Free kappa light chains b. Free lambda light chains IND, inflammatory neurological diseases; NIND, non-inflammatory neurological diseases and controls Ã The second best cut-off value in case the calculated cut-off resulted in 50% sensitivity or specificity that was considered as unacceptable. ÃÃ The second best cut-off value in case the calculated cut-off resulted in 50% specificity that was considered as unacceptable.