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Page 4 Bozzetti et al. Neuroimmunol Neuroinflammation 2021;8:1-13 I http://dx.doi.org/10.20517/2347-8659.2020.26

disability at baseline than at follow-up, recent studies indicate that serum NfL values could also be useful to
predict MS conversion in patients with a first demyelinating event, with lower NfL levels indicating reduced
[32]
risk of receiving a future MS diagnosis .

NfL IN RELAPSING-REMITTING MS
[33]
RRMS is the most common form of MS, involving 85% of affected patients . RRMS is characterized by
discrete and clearly definite attacks lasting days to weeks, followed by periods of partial/complete remission
[33]
in the absence of progressive clinical deterioration . Disease severity and lifelong prognosis of patients
with RRMS are highly variable, so that a correct subclassification of this condition according to risk of
[34]
future disease activity and final disability is of utmost importance to guide prompt therapeutic strategies .
The combination of clinical and radiological indicators of disease activity have long been used, despite the
high costs and incomplete predictive strength. To overcome these limitations and increase the sensitivity of
outcome prediction, the potential utility of different serum and CSF biomarkers have been explored over
the last years using high-sensitivity technology [20,25,35,36] . In particular, NfL levels have been analyzed and
[5]
compared to glial fibrillary acidic protein (GFAP) [25,36] , S100B, neuron-specific enolase (NSE) , chitinase
[25]
3-like 1 (CHI3L1) levels [20,25] , and to a panel of chemokines, matrix metalloproteinase-9, and ostepontin
with divergent data on the value of combining these biomarkers. The comparison between these biomarkers
and previously recognized clinical/radiological parameters of disability aimed to distinguish MS patients
and healthy controls, to improve the prediction of ongoing and future disease activity, to better predict
long-term outcome in terms of brain and spinal cord atrophy, final disability and risk of progression, and to
evaluate response to disease modifying therapy (DMT) .
[35]
The potential role of NfL in MS was proposed for the first time by Lycke et al. in 1998, who detected
[37]
increased levels of NfL in the CSF of RRMS patients in comparison with healthy controls. The authors
also demonstrated a significant correlation between NfL values and disability as assessed by EDSS score,
exacerbation rate, and time from last relapse. These findings gave important insight into MS pathogenetic
mechanisms, suggesting the presence of axonal damage in subjects with a relapsing-remitting course
[37]
and postulating a contribution of axonal pathology to disability . Subsequently, the potential utility of
measuring NfL levels in subjects with RRMS, monitoring longitudinal levels over time, offered indirect
cues to the understanding of NfL kinetics in blood and CSF [20,35,38-40] . Different studies displayed substantial
differences in terms of the matrix analyzed (serum, plasma, or CSF) and the performance of the assays used,
[41]
giving a clear spectrum of the evolution of the detection techniques and their related sensitivity . These
assays ranged from a second-generation ELISA [37,39,42-44] , to a third-generation electrochemiluminescence
technology [9,45] and, finally, to a fourth-generation SiMoA [9,20,25,38,40,46-48] that enables a reliable and highly
sensitive quantification and monitoring of serum/plasma NfL levels. In particular, the SiMoA novel
ultrasensitive technology increases the sensitivity of the assay allowing comparisons between pathological
and normal NfL values using small sample volume . This technical improvement, together with the
[22]
demonstration of a clear correlation between serum/plasma and CSF NfL levels [45,49] , now enables the
reliable measurement of NfL in blood samples, avoiding more costly and invasive procedures such as
lumbar puncture. This concept further supports the potential use of NfL as a promising biomarker useful
for longitudinal monitoring of disease activity and treatment response.

NfL values help to distinguish patients with RRMS from healthy controls
The first objective when investigating NfL levels in RRMS patients was to evaluate whether this biomarker
could be useful to differentiate patients from healthy controls. Significantly higher NfL levels in both
CSF [9,25,37,39,46,48] and serum [9,38,45,48] have been reported in patients vs. healthy controls using different
techniques. However, the substantial variation in NfL values observed in different studies prevented the
identification of a reproducible cut-off and suggested a great inter-individual variability, possibly influenced
by differences in measurement sensitivity, but not clearly related to demographic characteristics, sample