Review Article

Annals of Clinical Biochemistry

2017, Vol. 54(1) 5–13
! The Author(s) 2016
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DOI: 10.1177/0004563216652175
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Multiple sclerosis: assay of free immunoglobulin light

chains

DB Ramsden

Abstract
Over the past five years, a number of papers have appeared describing the assay of free immunoglobulin light chains in
cerebrospinal fluid to assist in the diagnosis of multiple sclerosis. The assay of kappa free immunoglobulin chains is being
advocated as a technically simpler and cheaper quantitative alternative to the qualitative detection of oligoclonal bands.
This article reviews the analytical and clinical characteristics of these immunoglobulin free light chain assays and places
them in their historical context and possible future developments.

Keywords
Analytical systems, clinical studies, immunoassay, laboratory methods, laboratory methods, neurological disorders
Accepted: 23rd April 2016

MS (SPMS). Most commonly, patients present with a

Introduction
In the last few years, a spate of papers has appeared con-
cerning the assay of free immunoglobulin light chains
(FLCs) in cerebrospinal fluid (CSF) for the diagnosis of
multiple sclerosis (MS). This review attempts to assess the
relevance of this methodology in the context of current
practice. MS is an autoimmune disease affecting approxi-
mately 150 per 100,000 in white Caucasians in developed
countries. The disease commonly presents in young adult-
hood/early middle age and adversely affects life expect-
ancy. The fundamental disease process is one of
demyelination of nerves in the central nervous system
and spinal cord. This is thought to arise from an imbal-
ance between activated effector and regulatory T cells;
however, B-cells are also involved.1 These, together with
weakening of the blood–brain barrier (BBB), contribute
to the presence of immunoglobulin molecules in the CSF.
The most common form of MS is where symptoms
appear for a time then decline – Relapsing – Remitting
MS (RRMS). Over time, this may progress to where
symptoms are continuous – secondary progressive
single incidence of an MS-like symptom, clinical isolated
syndrome (CIS). Many of these convert to clinically def-
inite MS (CDMS). Approximately 10% of patients, usu-
ally older patients, present with continuous manifestation
of the disease – primary progressive MS (PPMS).
The diagnosis and monitoring of MS pose a number
of problems. These include:
(a) differential diagnosis of MS;
(b) prediction of those patients with CIS who will con-
vert to CDMS;
(c) prediction of periods of relapse in RRMS;
(d) prediction of conversion of RRMS to SPMS.
Institute of Metabolism and Systems Research, The Medical School,
University of Birmingham, Birmingham, UK
Corresponding author:
DB Ramsden, Institute of Metabolism and Systems Research, The Medical
School, University of Birmingham, Birmingham B15 2TT, UK.
Email: D.B.Ramsden@bham.ac.uk
6 Annals of Clinical Biochemistry 54(1)
The only biochemical analysis widely used in MS
diagnosis historically is the detection of oligoclonal
immunoglobulin bands (OCBs); however, from the
beginning of the 21st century, the McDonald criteria
for MS diagnosis and its revisions have not included
OCB analysis.2–6 Nevertheless, OCB detection is still
frequently carried out, particularly where radiological
evidence is uncertain. Alongside the detection of OCBs,
estimates of the contribution of intrathecal synthesis of
immunoglobulin G (IgG) to total CSF IgG concentra-
tion have been advocated.7,8 Thus, OCB analysis,
together with intrathecal IgG synthesis quantification,
forms the current ‘gold standard’ biochemical method.
The methods described above have been centred on
the four-chain assembly that is the IgG molecule in
CSF. To drive efficient assembly of this molecule, one
of the two types of chain involved should be in excess.
Thermodynamics dictates that this is the smaller of the
two. Consequently, FLCs are synthesized in approxi-
mately 20% excess of heavy chains. The presence of j
and kFLCs in CSF has been recognized for almost
50 years.9 This has led to the development of a variety
assays for FLCs. From this body of work, the following
were determined:
(i) estimates of the CSF concentrations of the two
light chains,10–14
(ii) estimates of their intrathecal synthesis,12 and
(iii) the elevated concentrations of j and kFLCs in the
CSF of MS patients.12,15
Despite this work, compared with OCBs, little interest
was shown in the assay of FLCs in CSF as a diagnostic
and/or prognostic tool until recently. The current interest
in FLCs has been sparked by the availability of technically
simple assays for j and kFLCs in serum16 and the under-
standing of the significance of the data generated for the
management of myeloma and other conditions.17–19
CSF FLC assays and analytical sensitivity
Current assays have analytical sensitivities which cover
at least the upper segment of the ‘normal’ control
ranges of j and kFLC concentrations in CSF
(Table 1).20,21 The assays used are based on antibodies
against epitopes on the FLCs that are covered in the
four-chain assembly that forms the IgG structure. The
Binding Site assay uses polyclonal antibodies against
these epitopes, whereas the assays from three other
companies use monoclonal antibodies. The Binding
Site and Siemens assays are nephelometric assays,
which typically have the anti-FLC antibody on latex
particles. The assay may be carried out on automated
nephelometers, allowing multiple samples to be ana-
lysed in a short time. The Biovendor and Polygnost
assays are typical non-competitive enzyme-linked
immunosorbent assays (ELISAs) in which the specific
anti-FLC antibody is the initial capture antibody
coated on the bottom of the wells in a 96-well plate.
There are no data from a single laboratory that directly
compare the performances of the different assays, but
in what may be considered an aside, Makshakov et al.22
indicate that their ELISA performed better than a
nephelometric assay. Further details of the assays are
available on the manufacturers’ websites, listed at the
end of the reference section.
Typical lower limits of detection quoted by
authors are:
(a) <0.1 mg/L for both j and kFLCs,21
(b) 0.06 mg/L for jFLC and 0.08 mg/L for kFLC,
with intra-assay coefficient of variation of 4%
and 4.8% at 0.54 mg/L and inter-assay CV of
14% and 7.45% at 0.55 mg/L for jFLC and
kFLC, respectively.23,24
Analytes, analyte structure and metrics
The ability to quantify FLCs raises the question as to
what analyte(s) is (are) to be assayed, jFLC only or j
and kFLCs, and how is (are) this (these) to be employed.
There is universal agreement that jFLC concentration is
elevated in CSF from CDMS patients and thus is the
more important analyte. Some authors do not assay
kFLC concentration. Those who do assay kFLC concen-
trations report elevated concentrations in MS CSF cf
those in CSF from patients with non-infective neuro-
logical diseases, although the relative increase is less
than that of jFLC concentration in MS.11,22 In patients
with infectious neurological diseases, the increase in CSF
kFLC concentration is of similar magnitude to or greater
than that of jFLC concentration in MS.
Mass spectroscopy analysis of MS CSF showed
jFLCs were present as both monomers and dimers,
whereas kFLCs were present primarily as dimers.24
How jFLC dimers and monomers influence the result
in assays is unclear. For example, in nephelometric
assays, where the rate of aggregation of antibody and
antigen is the basis of the assay, does one jFLC dimer
bind one or two antibody molecules? Although this may
seem esoteric, the monomer:dimer could affect the result
in borderline cases. No current literature addresses this
question. The other result from this analysis is surpris-
ing, in that out of 56 patients’ CSF, five were found to
have no elevation of jFLC concentration but increased
kFLC concentration. This is a high percentage and con-
trasts with the results of other recent authors who
assayed both forms of light chain, but is in agreement
with the work of Bracco et al.10
Ramsden 7

Table 1. Concentration of kappa free immunoglobulin light chains in neurological disease.

Number Median Assay Antibody type

Authors Subject group of subjects or meana Range method (assay source)

Hasan Smith et al.22 NNI 90 0.14 0.06–0.79 Neph Polyclonal (TBS)

NI 17 0.25 0.06–4.47
RRMS 21 8.01 0.14–29.12
CIS 21 8.08 0.06–26.03
Senel et al.20 NNI 77 0.013 0.004 -0.025 ELISA Monoclonal (BioVendor)
NB 17 1.5 0.64–2.24
CDMS 20 0.62 0.38–1.34
CIS 77 0.13 0.014–0.72
Duranti et al.56 NNI 33 0.07 0.04–0.33 Neph Monoclonal (Siemens)
NI 24 0.095 0.02–2.31
CDMS 23 1.08 0.14–11.7
Arneth and Birklein25 NNI 42 0.43a 0.05–4.59 Neph Polyclonal (TBS)
NI 24 4.4a 0.06–28.3
CDMS 44 4.3a 0.38–27.1
Presslauer et al.30 Control 45 0.18 0.13–0.22 Neph Polyclonal (TBS)
Encep/Men 41 0.55 0.32–1.27
CDMS 70 4.62 1.41–10
CIS 29 2.95 1.39–8.65
Desplat-jégo et al.21 NNI 33 0.9 0.03–2.91 Neph Polyclonal (TBS)
8 1.32 0.49–12.8
CDMS 33 1.26 0.33–14.6
CIS 15 1.18 0.28–6.89
Makshakov et al.51 NNI 43 0.05 0.032–0.076 ELISA Monoclonal (PLSP)
NI 16 0.45 0.189–0.59
CIS-MS 98 0.45 0.225–0.965
CIS-NMS 41 0.11 0.004–0.27
CDMS: clinically definite multiple sclerosis; CIS: clinical isolated syndrome; CIS-MS: clinical isolated syndrome converting to multiple sclerosis within 2
years; CIS-NMS Clinical isolated syndrome not converting to multiple sclerosis within 2 years; Encep/Men: encephalitis and meningitis; Neph:
nephelometry; NB: neuroborreliosis; NNI: non-inflammatory neurological disease, NI inflammatory neurological disease other than MS; RRMS:
relapsing – remitting multiple sclerosis; TBS: the binding site; PLSP: Polignost Ltd.
a
Mean.

Metrics used to assess clinical relevance include:
(a) CSF FLC Concentration CSF total protein ratio
½CSF FLC:½CSF protein23
(b) FLC Quotient (QFLC),20,22,25,26
QFLC ¼ ½CSF kFLC=½serum kFLC
(c) jFLC Index, an estimate of jFLC intrathecal
synthesis,21
(d) Another estimate of intrathecal synthesis
obtained from plots of CSF QFLC against
(CSF albumin)/(serum albumin) (Qalb) using a
dividing line described by equation (4a)
ð1Þ which gives the theoretical contribution of FLCs
diffusing from the blood into the CSF across
the BBB to CSF total (FLC) – theoretical QFLC
(Theor QFLC) – separating patients displaying intra-
ð2Þ thecal synthesis (above the line) from ones not
doing so25,26
Theor QFLC ¼ a=ðb sqrtðQ2alb  b2 Þ  cÞ ð4aÞ

kFLC Index ¼ ð½CSF kFLC=½serum kFLCÞ= where a, b and c are arbitrary constants
ð½CSF Albumin=½serum AlbuminÞ (e) A modified version of (d) using equation (4b),
which takes into account the fact that
ð3Þ jFLCshave a lower molecular weight than
8 Annals of Clinical Biochemistry 54(1)
albumin,27
Theor QFLC ¼ b sqrtðððQalb þ cÞ=aÞ2  1Þ ð4bÞ
a, b and c are empirical constants
(f) Intrathecal FLC fraction (jFLCIF), a fourth esti-
mate of intrathecal synthesis.28 This method first
estimates the theoretical upper limit of jFLC dif-
fusion through the BBB (jFLCLim) by reference to
albumin diffusion
kFLCLim ¼ 0:9358  QAlb0:6687 ð5aÞ
then calculates local jFLC synthesis (jFLCLoc) by
subtracting the theoretical from the actual CSF
concentration,
kFLCLoc ¼ ðkFLCRatio  kFLCLim Þ  kFLCSerum
ð5bÞ
and finally expresses the estimate of intrathecal
synthesis as a percentage of total CSF jFLC con-
centration (jFLCIF)
kFLCIF ¼ ðkFLCLoc =kFLCCSF Þ  100 ð5cÞ
The disproportionate elevation of CSF jFLC con-
centration cf kFLC concentration is the most common
phenomenon in CDMS and CIS cases (Table 1), but
there is no agreement as to how this should be used.
Examples of the use of each metric are present in the
current literature. Zeman et al.27 surveyed numerous
metrics and found that several of them, including
simple ones were good estimates of intrathecal synthe-
sis. Although it may be argued that considering
whether CSF jFLC concentration is elevated without
considering intrathecal synthesis does not in itself allow
one to differentiate among causes of the increase, this
logic is equally true if estimates of intrathecal synthesis
alone are considered. Thus, in practical terms, the
simplest metric may be all that is required to confirm
diagnosis of MS in the majority of instances, because of
the diverse clinical picture between MS and other dis-
eases with elevated FLCs and OCBs. Elevated kFLCs
may be considered chiefly as marker of infection.
Nevertheless, in cases where the diagnosis of MS is
unclear, kFLC concentrations may need to be taken
into account.29 Further consideration regarding the
choice of metric is given in the Conclusions section.
Clinical sensitivity and specificity and
differential diagnosis of MS
The first clinical objective in assessing the worth of
FLC assays is to distinguish patients with CDMS
from patients with (a) other neurological diseases, (b)
other neurological inflammatory diseases and (c)
non-neurological non-inflammatory disease. Table 1
summarizes the results from seven groups of authors.
A quite wide range of values is quoted, even for the
control groups (neurological conditions – non-inflam-
matory) and the CDMS groups. These two patient
groups are the easiest to compare because they are
the most clinically homogeneous. The high jFLC con-
centrations in CSF in the CDMS groups are the dom-
inant feature. An elevated jFLC concentration is not
unique to MS; however, as Presslauer et al.30 pointed
out, most of the other diseases that do present with a
high CSF jFLC concentration have dissimilar clinical
characteristics to MS.
The CIS groups have varying numbers of patients
who progress to CDMS and the inflammatory non-
MS, neurology disease groups in the majority of cases
are composed of varying numbers of different diseases.
These facts probably account in large part for the dis-
parity in levels reported by the different authors. As a
consequence of this diverse nature, half the reports
show the median (or mean) jFLC concentration of
the inflammatory, non-MS neurology disease group to
be notably lower than that of the MS group, with the
other half reporting values very similar to or higher
than that of the MS group. Only Senel et al.20 and
Presslauer et al.30 have sufficient numbers to segregate
the inflammatory non-MS, neurology disease group
into individual diseases, jFLC concentration being
higher in neuroborreliosis20 and lower in encephalitis/
meningitis30 cf CDMS.
Table 2 summarizes clinical sensitivity and specificity
reported by authors who used different metrics. All sup-
port the concept that a high value for the relevant
jFLC metric supports a diagnosis of MS, assuming
the appropriate clinical context. This contention is
strengthened by the findings of the latest, multicentre
trial.28 Furthermore, almost all recent authors claim
that, for MS diagnosis, their jFLC metric has sensitiv-
ity and specificity which are equal to or better than that
using the OCB ‘gold standard’. An illustration of the
potentially greater sensitivity of FLC assays cf OCB
assays is the account by Goffette et al.31 who reported
the detection of jFLCs in the absence of OCBs.
A minority of authors claims that a k index adds
extra definition to the sensitivity,29 whereas the major-
ity does not. Given that the numbers of cases reported
are small, this latter point awaits resolution.
Prognostic value of free light chain
metrics: CIS conversion
Prediction of CIS conversion to CDMS is the second
major objective of CSF assays, but the literature on
Ramsden 9

Table 2. Clinical sensitivity and specificity of CSF free immunoglobulin light chain assay in multiple
sclerosis.

References Metric Cut-off Sensitivity Specificity, %

Presslauer et al.28 Kappa index 5.9 95% 95

Hasan-Smith et al.23 [Kappa] 0.87mg/L 96.2% 98.1
[Lambda] 0.57 mg/L 60.4% 98.1
[Kappa] þ [lambda] 1.47 mg/L 96.2% 98.1
[Kappa]/[Lambda] 0.975 94.2% 98.1
Bernadi et al.30 [Kappa] 0.56 mg/L
Kappa index 7.82 95%
[Lambda] 0.31 mg/L
Lambda index 4.36 83%
Kappa and lambda indices 100%
Duranti et al.56 Kappa index 12 95% 91
Desplat-jégo et al.21 Kappa index 20 69.7% 81.8
Makshakov et al.22a [Kappa] >0.103 mg/L 92.9 88.8
QFLC >0.0083 88.24 89.5
Fischer et al.25b [Kappa] 0.5 mg/L 93.8%
[x]: concentration of variable x.
a
Determined by ROC analysis between CIS-MS and control group.
b
Demonstration of intrathecal kappa FLC synthesis in MS.

FLC metrics relating to conversion CIS to CDMS is
comparatively scarce; therefore, it is relevant to con-
sider the recent literature on OCB analysis. Within
the past three years, three major studies have been
reported. Dobson et al.32 described a meta-analysis of
71 scientific papers covering 2685 CIS patients of whom
1841 were OCB positive and 844 OCB negative. From
these data, it was concluded that OCB-positive CIS
patients had an odds ratio of 9.88 for conversion to
CDMS. Subsequently, Kuhle et al.33 with data from
1047 CIS patients (conversion to CDMS 59.5%) and
Tintore et al.34 with data from 792 CIS patients (con-
version to CDMS 57.2%) concluded that the presence
of OCBs was a highly significant prognostic factor.
Given that OCBs and FLCs arise from B-cell activ-
ity, it would seem logical to assume that FLC metrics
would have a similar ability compared with that of
either OCBs or estimates of intrathecal IgG synthesis
to predict the conversion of CIS to CDMS. This con-
cept is supported by the data of Zeman et al.20 who
assessed an estimate of oligoclonal IgG synthesis com-
pared with a number of FLC metrics in 75 patients.
They concluded that CSF jFLC was a reliable pre-
dictor of intrathecal synthesis. Villar et al.35 compared
the CSF jFLC concentration of 78 CIS patients – 49
with CSF jFLC concentration above a cut-off of two
standard deviations above the CIS mean plus 2 SD of
control group values against those with concentrations
below this. The group with the higher concentrations
had a higher probability of conversion to CDMS
(P < 0.0001). After follow-up, most patients with high
CSF jFLC concentration were found to have con-
verted to CDMS, whereas more than 70% of those
with the lower concentration remained as CIS.
‘Univariate analysis gave a hazard ratio of 9.13 (95%
CI¼2.8–29.78). These results were confirmed by multi-
variate analysis adjusted for sex, age and basal MRI
findings, where the hazard ratio was 6.41; (95%
CI¼1.88–162 21.78) (P < 0.003)’.35
Senel et al. followed 77 CIS patients for two years.
They reported that the kappa quotient (equation (2))
was higher in CIS patients (n ¼ 39) who progressed to
CDMS than in those who did not (n ¼ 38). They found
that 86.8% of CIS patients who converted to MS had
elevated kappa quotients, compared with only 61.5%
of CIS patients who did not convert (P < 0.001).
They proposed that these data are suggestive of the
prognostic value of their kappa metric. Nevertheless,
when they compared OCB analysis with the kappa
quotient, their results were: for the kappa quotient –
sensitivity 86.8%; specificity 38.5%; positive predictive
value (PPV) 57.9%; negative positive predictive value
(NPV) 75%, and for OCB analysis – sensitivity 89.1%;
specificity 33.3%; PPV 57.4%; NPV 81.3%. They con-
cluded that the kappa quotient was of similar value but
not superior to OCB analysis in predicting conversion
of CIS to CDMS.20 Further support for the fact that a
high jFLC metric is predictive of CIS conversion to
CDMS comes from the report of Makshakov et al.22
who followed 141 patients initially presenting with CIS.
10
Of these, 98 had converted to CDMS by the end of the
second year. The group of patients who converted to
CDMS had a higher median CSF kappa concentration
(0.45 mg/L, range: 0.225–0.965; P < 0.0001) compared
with that of those who remained clinically stable
(0.11mg/L, range 0.004–0.27).22 In contrast to the sug-
gestion of a positive predictive power for jFLC met-
rics, Desplat-jégo et al. reached the opposite
conclusion. They followed 12 CIS patients over a
period of 12 months and correlated CSF kappa index
with:
(a) rate of CIS conversion to CDMS according
McDonald’s criteria;
(b) time to conversion to CDMS according
McDonald’s criteria;
(c) number of relapses;
(d) number of relapses/length of follow-up;
(e) initial and final EDSS score.
They found no correlation between the jFLC index
and these variables.21 Presslauer et al.36 also claim there
is no significant difference between the jFLC index of
the CIS group converting to MS (mean ¼ 77.8) com-
pared with the stable CIS group (mean ¼ 82.7).
Prognostic value of free light chain
metrics: CDMS progression
Unlike the value of OCB analysis for predicting
conversion CIS to CDMS, the prognostic value of
any CSF variable to predict progression of the disease
once established is open to question. Becker et al.,37
after assessing the results of 409 patients followed up
over 4.5 years concluded that none of 11 clinical and
biochemical parameters predicted disease progress.
These included OCBs and IgG index. Similarly,
Anagnostouli et al.38 assessed 108 MS patients, com-
paring dysfunction with OCB concentration. They con-
cluded that the presence of CSF OCBs in their MS
patients tended to be related to widespread cognitive
changes, specifically worse visual memory (Rey’s com-
plex figure test-recall; P ¼ 0.006). They proposed that
OCB analysis as a prognostic factor needs further study
before any firm conclusion can be drawn with regards
to its value.
In contrast to the negative opinions cited above
regarding OCB analysis, early data on FLCs are more
positive. Rudick et al.39 studied 36 patients over a
median follow-up time of 38.9 months. They evaluated
myelin basic protein, IgG synthesis rate, IgG index and
j and kFLCs as prognostic factors. Expanded disability
status scale, the Ambulation Index, the 9 Hole Peg Test
and the Box and Blocks test were used to assess the
disease progression. They concluded that CSF jFLC
Annals of Clinical Biochemistry 54(1)
concentrations were the best predictor of physical
deterioration.39 Subsequently, Rinker et al., studied
57 CDMS patients (RRMS 23; SPMS 28; PPMS 6)
with a median disease duration of 15 years (range 1
to 30 years). Patients were divided into one group
with CSF jFLC concentration 5 1.53 mg/L and
another with <1.53 mg/L. The high CSF jFLC concen-
tration group had a higher likelihood of a more severe
disease course than the low concentration group. This
was evidenced by a high likelihood of requiring ambu-
latory assistance within 10 years and over the disease
course. The group also had greater disability relative to
disease duration, as estimated by a multiple sclerosis
severity score. They reported that the PPV of CSF
jFLC concentration 5 1.53 mg/L was:
(a) for requiring ambulatory assistance within 10
years – 66.7%;
(b) for requiring ambulatory assistance over the
course of the disease – 88.9%;
(c) for the likelihood of reaching a multiple sclerosis
severity score >6 was 88.9% with 95%
confidence.40
These findings contrast with those of Desplat-jégo
et al. who followed a group of 15 CIS patients convert-
ing to CDMS. They found no correlation between
jFLC indices and disease progression as judged by:
(i) time to conversion to MS according McDonald’s
criteria,
(ii) number of relapses,
(iii) number of relapses/length of follow-up,
(iv) initial and final EDSS score.21
The findings of Desplat-jégo et al. are in accord with
the results of the multicentre Avonex phase III trial
described by Rudick et al. in which CSF jFLC concen-
tration did not change significantly in either treatment
or placebo groups over the two year period of the
trial.41 Thus, CSF jFLC concentration did not reflect
improvement in the clinical status of patients following
treatment with the drug. Nevertheless, CSF jFLC con-
centration was weakly correlated with Gd lesion
volume and T2 lesion volume.
Conclusions
Much interest has been generated by the introduction
of technically simple, rapid, quantitative assays for
determination of CSF j and kFLC concentrations,
allowing the possibility of replacing the technically
more difficult, time-consuming, qualitative assay of
OCBs still used in many hospitals for the diagnosis of
MS. In turn, the use of these assays has opened up the
Ramsden
question of what FLC metric to use. The answer
depends in part on the question of why the metric is
being used – diagnosis of MS or prognosis of conver-
sion of CIS to CDMS. As can be seen above, groups of
authors have used a variety of metrics, primarily based
on the jFLC concentration. No consensus has as yet
been arrived at as to which to use. Applying the philo-
sophical principle advocated by William of Ockham,
commonly known as Occam’s razor, proposes that
when faced with a number of options of indeterminate
certainty, often the choice of the option with least com-
plexity is the best. This principal suggests that in the
majority of cases it would seem unlikely that the more
complex metrics are necessary for a diagnosis of MS to
be made. This idea is in accord with the data of Zeman
et al.42 who surveyed numerous metrics using the same
patients’ serum samples and CSF. They found that sev-
eral of the metrics, including simple ones such as CSF
concentration, were good estimates of intrathecal syn-
thesis. Interestingly, Bonnan points out that none of the
estimates of intrathecal IgG synthesis are truly accur-
ate, with the best having possibly up to an error of
20%.43 Similarly, estimates of intrathecal FLC synthe-
sis, such as kappa index, by definition must be less
technically accurate than kappa concentration, because
they contain the errors of this parameter plus those of
the estimates of serum kappa and albumin and CSF
albumin concentrations. Zeman et al.42 also commented
on the lack of an efficient means of estimating intrathecal
kappa synthesis when they proposed their modified for-
mula (equation (4b))42 rather than using for intrathecal
FLC synthesis the equation originally proposed by
Reiber et al. for IgG synthesis.44 Given that:
(a) in molecular terms light chains are synthesized at
more than twice the rate of the fully formed IgG
and approximately 20% greater than individual
heavy chains,
(b) IgA and IgM may also be formed by intrathecal
synthesis, the synthesis of which further contribute
to total concentration of FLCs in the CSF because
of the necessity to synthesize an excess of light
chains compared with that of heavy chains, and
(c) the molecular weight of the light chain used to
derive formulae such as that proposed by Zeman
et al.42 should take into account the mono-
mer:dimer, estimates of total kappa light chain
synthesis (free and incorporated into Ig molecules)
may give the clearest picture of B-cell activity.
Nevertheless, such a detailed set of analyses is unli-
kely to be carried out in general practice.
Further limited support for the idea that the sim-
plest metric may be all that is necessary comes
from Makshakov et al.22 These authors compared
total CSF kappa concentration with kappa
11
quotient for the prediction of conversion of CIS
to CDMS and found that the former was a better
predictor. Nevertheless, because the diagnosis of
MS is such an overwhelmingly important event,
it may be necessary to consider estimates of intra-
thecal synthesis as well as kappa concentration,
both being viewed in the context of other biochem-
ical and clinical parameters. All of the above
emphasize the fact that consensus guidelines
based on data for a large group of patients are
required to indicate which iFLC metric or com-
bination of metrics, is the optimum. Possibly these
guidelines should include the consideration of
kFLC concentration where the results of MRI
and the chosen kappa metric are inconclusive.
Despite this area of uncertainty, the properties of
FLC assay make it an attractive option for CSF
analysis in the short to medium term.
Thus said, although there is much recent interest in
FLC assay, the results generated have an air of déjà-vu.
Much of what is now being reported has been reported
before. Examples of this are the recognition of the pre-
dominance of the increased CSF jFLC concentration
cf kFLC concentration and the near equality or super-
iority of kappa metrics compared with OCB analysis in
the diagnosis of MS. Twenty-seven years ago Rudick
et al. wrote, ‘Our results strongly suggest that free kappa
light chains in CSF are the single best quantitative assay
to support a clinical diagnosis of MS’.45 Whether CSF
FLC quantification has any long-term prognostic value
is more doubtful. It is in most instances a one-off assay,
giving a unique insight into B-cell activity at that time.
The likelihood of a second CSF sample being taken is
small. If the level of B-cell activity does not change with
time, as asserted for CSF jFLC concentration by
Rudick et al.,42 although symptoms might improve fol-
lowing treatment or spontaneous remission, what rele-
vance can CSF jFLC assay have for prognosis once the
disease is established. Interestingly, the report of Mehta
et al.46 that urinary excretion of jFLC increased in
periods of relapse has never been replicated, for what-
ever reason. Nonetheless, if this were true, and given we
now have the ability to determine the primary sequence
of proteins with relative ease, it may be possible to
monitor the concentrations of intrathecally produced
FLCs specific to the individual patient in an easily
obtainable fluid. This would be a sophisticated vari-
ation on the theme of detection of measles, rubella
and varicella zoster antibodies, which can be detected
in a majority of MS patients.47 Nevertheless, until a
widely accepted serum or urine biomarker is developed,
the absence of which has been illustrated in the recent
reviews of Housey et al.48 and D’Ambrosio et al.49,
emphasis on MRI results in any subsequent modification
12
of the McDonald guidelines will remain. This is espe-
cially the case because in time there will be increased
confidence in MRI diagnostic and prognostic protocols
in recently issued guidelines by members of the
MAGNIMS group.5,6,50–54 Further, improvements are
being made in the ability of software to analyse the ensu-
ing results, such as that described by Abbasian Ardakani
et al. which reveals details not visible to the human eye.55
Websites of commercial sources
of FLC assays
Siemens: http://www.healthcare.siemens.co.uk/plasma-
protein/assays/n-kappa-and-lambda-assays
The Binding Site Ltd: http://www.bindingsite.com/en/
discover/freelite-and-hevylite
Biovendor: https://www.biovendor.com/country1/pro
duct/immunoassays/immunoglobulin-free-light-chains-
kappa-and-lambda-human-elisa
Polygnost St Petersburg: http://all-russian-business.
com/company/981900
Declaration of conflicting interests
Ad hoc consultation for The Binding Site Ltd., since the company’s foundation.
Funding
The author(s) received no financial support for the research, authorship, and/or
publication of this article. Following acceptance by ACB the author has has
applied for and received a bursary from The Binding Site Ltd.
Ethical approval
Not applicable.
Guarantor
DBR.
Contributorship
DBR sole author.
References
1. Sandberg-Wollheim M, Zettervall O, et al. In vitro synthesis of IgG by cells
from the cerebrospinal fluid in a patient with multiple sclerosis. Clin Exp
Immunol 1969; 4: 401–405.
2. McDonald WI, Compston A, Edan G, et al. Recommended diagnostic cri-
teria for multiple sclerosis: guidelines from the International Panel on the
diagnosis of multiple sclerosis. Ann Neurol 2001; 50: 121–127.
3. Polman CH, Reingold SC, Edan G, et al. Diagnostic criteria for multiple
sclerosis: 2005 revisions to the ‘‘McDonald Criteria.’’ Ann Neurol 2005; 58:
840–846.
4. Polman CH, Reingold SC, Banwell B, et al. Diagnostic criteria for multiple
sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol 2011; 69:
292–302.
5. Rovira À, Wattjes MP, Tintoré M, et al. Evidence-based guidelines:
MAGNIMS consensus guidelines on the use of MRI in multiple sclerosis-
clinical implementation in the diagnostic process. Nat Rev Neurol 2015; 11:
471–482.
6. MAGNIMS study group; Wattjes MP, Rovira À, et al. Evidence-based
guidelines: MAGNIMS consensus guidelines on the use of MRI in multiple
sclerosis-establishing disease prognosis and monitoring patients. Nat Rev
Neurol 2015; 11: 597–606.
Annals of Clinical Biochemistry 54(1)
7. Reiber H and Felgenhauer K. Protein transfer at the blood cerebrospinal
fluid barrier and the quantitation of the humoral immune response within
the central nervous system. Clin Chim Acta 1987; 163: 319–328.
8. Reiber H, Ungefehr S and Jacobi C. The intrathecal, polyspecific and oligo-
clonal immune response in multiple sclerosis. Mult Scler 1998; 4: 111–117.
9. Link H. Immunoglobulin G and low molecular weight proteins in human
cerebrospinal fluid. Chemical and immunological characterization with
special reference to multiple sclerosis. Acta Neurol Scand 1967; 43(Suppl
28): l–136.
10. Bracco F, Gallo P, Menna R, et al. Free light chains in the CSF in multiple
sclerosis. J Neurol 1987; 234: 303–307.
11. DeCarli C, Menegus MA and Rudick RA. Free light chains in multiple
sclerosis and infections of the CNS. Neurol 1987; 37: 1334–1338.
12. Fagnart OC, Sindic CJM and Laterre C. Free kappa and lambda light
chain levels in the cerebrospinal fluid of patients with multiple sclerosis
and other neurological diseases. J Neuroimmunol 1988; 19: 119–132.
13. Stanescu GL, Swick AR, Tuohy VK, et al. Sensitive competitive-binding
ELISAs for quantifying free kappa and lambda light chains in cerebro-
spinal fluid. J Clin Lab Anal 1991; 5: 206–211.
14. Rudick RA, Pallant A, Bidlack JM, et al. Free kappa light chains in mul-
tiple sclerosis spinal fluid. Ann Neurol 1986; 20: 63–69.
15. Lolli F, Siracusa G, Amato MP, et al. Intrathecal synthesis of free
immunoglobulin light chains and IgM in initial multiple sclerosis. Acta
Neurol Scand 1991; 83: 239–243.
16. Bradwell AR, Carr-Smith HD, Mead GP, et al. Highly sensitive, auto-
mated immunoassay for immunoglobulin free light chains in serum and
urine. Clin Chem 2001; 47: 673–680.
17. Drayson M, Tang LX, Drew R, et al. Serum free light-chain measurements
for identifying and monitoring patients with nonsecretory multiple mye-
loma. Blood 2001; 97: 2900–2902.
18. Abraham RS, Katzmann JA, Clark RJ, et al. Quantitative analysis of
serum free light chains. A new marker for the diagnostic evaluation of
primary systemic amyloidosis. Am J Clin Pathol 2003; 119: 274–278.
19. Hutchison CA, Burmeister A, Harding SJ, et al. Serum polyclonal
immunoglobulin free light chain levels predict mortality in people with
chronic kidney disease. Mayo Clin Proc 2014; 89: 615–622.
20. Senel M, Tumani H, Lauda F, et al. Cerebrospinal fluid immunoglobulin
kappa light chain in clinically isolated syndrome and multiple sclerosis.
Plos One 2014; 9: 1–7.
21. Desplat-jégo S, Feuillet L, Pelletier J, et al. Quantification of immuno-
globulin free light chains in cerebrospinal fluid by nephelometry. J Clin
Immunol 2005; 25: 338–345.
22. Makshakov G, Nazarov V, Kochetova O, et al. Diagnostic and prognostic
value of the cerebrospinal fluid concentration of immunoglobulin free light
chains in clinically isolated syndrome with conversion to multiple sclerosis.
PLoS One 2015; 10: e0143375.
23. Hassan-Smith G, Durant L, Tsentemeidou A, et al. High sensitivity and
specificity of elevated cerebrospinal fluid kappa free light chains in sus-
pected multiple sclerosis. J Neuroimmunol 2014; 276: 175–179.
24. Kaplan B, Golderman S, Yahalom G, et al. Free light chain monomer-
dimer patterns in the diagnosis of multiple sclerosis. J Immunol Methods
2013; 390: 74–80.
25. Fischer C, Arneth B, Koehler J, et al. Kappa free light chains in cerebro-
spinal fluid as markers of intrathecal immunoglobulin synthesis. Clin Chem
2004; 50: 1809–1813.
26. Arneth B and Birklein F. High sensitivity of free lambda and free kappa
light chains for detection of intrathecal immunoglobulin synthesis in cere-
brospinal fluid. Acta Neurol Scand 2009; 119: 39–44.
27. Zeman D, Kušnierová P, Bartoš V, et al. Quantitation of free light chains
in the cerebrospinal fluid reliably predicts their intrathecal synthesis. Ann
Clin Biochem 2016; 53: 174–176.
28. Presslauer S, Milosavljevic D, Hübl W, et al. Validation of kappa free light
chains as a diagnostic biomarker in multiple sclerosis and clinically isolated
syndrome: a multicenter study. Mult Scler 2015; 22: 502–510.
29. Bernardi G and Cataldo I. La determinazione delle catene leggere libere nel
liquido cefalorachidiano: l’esperienza di due laboratori Italiani. Biochim
Clin 2013; 37: 389–394.
30. Presslauer S, Milosavljevic D, Brücke T, et al. Elevated levels of kappa free
light chains in CSF support the diagnosis of multiple sclerosis. J Neurol
2008; 255: 1508–1514.
31. Goffette S, Schluep M, Henry H, et al. Detection of oligoclonal free kappa
chains in the absence of oligoclonal IgG in the CSF of patients with sus-
pected multiple sclerosis. J Neurol Neurosurg Psychiatry 2004; 75: 308–310.
Ramsden
32. Dobson R, Ramagopalan S, Davis A, et al. Cerebrospinal fluid oligoclonal
bands in multiple sclerosis and clinically isolated syndromes: a meta-ana-
lysis of prevalence, prognosis and effect of latitude. J Neurol Neurosurg
Psychiatry 2013; 84: 909–914.
33. Kuhle J, Hardmeier M, Disanto G, et al. European long-term follow-up
study group in interferon b-1b in secondary-progressive multiple sclerosis.
A 10-year follow-up of the European multicenter trial of interferon b-1b in
secondary-progressive multiple sclerosis. Mult Scler 2015; 22: 533–543.
34. Tintore M, Rovira A, Rıo J, et al. Defining high, medium and low impact
prognostic factors for developing multiple sclerosis. Brain 2015; 138:
1863–1874.
35. Villar LM, Espiño M, Costa-Frossard L, et al. High levels of cerebrospinal
fluid free kappa chains predict conversion to multiple sclerosis. Clin Chim
Acta 2012; 413: 1813–1816.
36. Presslauer S, Milosavljevic D, Hübl W, et al. Kappa free light chains:
diagnostic and prognostic relevance in MS and CIS. Plos One 2014; 9: 1–6.
37. Becker M, Latarche C, Roman E, et al. No prognostic value of routine
cerebrospinal fluid biomarkers in a population-based cohort of 407 mul-
tiple sclerosis patients. BMC Neurol 2015; 15: 79.
38. Anagnostouli M, Christidi F, Zalonis I, et al. Clinical and cognitive impli-
cations of cerebrospinal fluid oligoclonal bands in multiple sclerosis
patients. Neurol Sci 2015; 36: 2053–2060.
39. Rudick RA, Medendorp SV, Namey M, et al. Multiple sclerosis progres-
sion in a natural history study: predictive value of cerebrospinal fluid free
kappa light chains. Mult Scler 1995; 1: 150–155.
40. Rinker JR II, Trinkaus K and Cross AH. Elevated CSF free kappa light
chains correlate with disability prognosis in multiple sclerosis. Neurology
2006; 67: 1288–1290.
41. Rudick RA, Cookfair DL, Simonian NA, et al. Cerebrospinal fluid
abnormalities in a phase III trial of Avonex IFNb-1a for relapsing multiple
sclerosis. J Neuroimmunol 1999; 93: 8–14.
42. Zemana D, Hradilek P, Kusnierova P, et al. Oligoclonal free light chains in
cerebrospinal fluid as markers of intrathecal inflammation. Comparison
with oligoclonal IgG. Biomed Pap Med Fac Univ Palacky Olomouc Czech
Repub 2015; 15: 104–113.
43. Bonnan M. Intrathecal IgG synthesis: a resistant and valuable target for
future multiple sclerosis treatments. Mult Scler Int 2015; 2015: 296184.
13
44. Reiber H. Proteins in cerebrospinal fluid and blood: barriers, CSF flow rate
and source-related dynamics. Restor Neurol Neurosci 2003; 21: 79–96.
45. Rudick RA, French CA, Breton D, et al. Relative diagnostic value of
cerebrospinal fluid kappa chains in MS: comparison with other immuno-
globulin tests. Neurology 1989; 39: 964–968.
46. Mehta PD, Cook SD, Troiano RA, et al. Increased free light chains in the
urine from patients with multiple sclerosis. Neurology 1991; 41: 540–544.
47. Reiber H, Ungefehr S and Jacobi C. The intrathecal, polyspecific and
oligoclonal immune response in multiple sclerosis. Mult Scler 1998; 4:
111–117.
48. Housley WJ, Pitt D and Hafler DA. Biomarkers in multiple sclerosis. Clin
Immunol 2015; 161: 51–58.
49. D’Ambrosio A, Pontecorvo S, Colasanti T, et al. Peripheral blood bio-
markers in multiple sclerosis. Autoimmun Rev 2015; 14: 1097–1110.
50. Giorgio A, Battaglini M, Rocca MA, et al. Location of brain lesions pre-
dicts conversion of clinically isolated syndromes to multiple sclerosis.
Neurology 2013; 80: 234–241.
51. Popescu V, Agosta F, Hulst HE, et al. Brain atrophy and lesion load pre-
dict long term disability in multiple sclerosis. J Neurol Neurosurg
Psychiatry 2013; 84: 1082–1091.
52. Gass A, Rocca MA, Agosta F, et al. MRI monitoring of pathological
changes in the spinal cord in patients with multiple sclerosis. Lancet
Neurol 2015; 14: 443–454.
53. Rocca MA, Amato MP, De Stefano N, et al. Clinical and imaging assess-
ment of cognitive dysfunction in multiple sclerosis. Lancet Neurol 2015; 14:
302–317.
54. Vrenken H, Jenkinson M, Horsfield MA, et al. Recommendations to
improve imaging and analysis of brain lesion load and atrophy in longitu-
dinal studies of multiple sclerosis. J Neurol 2013; 260: 2458–2471.
55. Abbasian Ardakani A, Gharbali A, Saniei Y, et al. Application of texture
analysis in diagnosis of multiple sclerosis by magnetic resonance imaging.
Glob J Health Sci 2015; 7: 68–78.
56. Duranti F, Pieri M, Centonze D, et al. Determination of jFLC and j Index
in cerebrospinal fluid: a valid alternative to assess intrathecal immuno-
globulin synthesis. J Neuroimmunol 2013; 263: 116–120.