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Table 2 Data on longitudinal behaviour of NFs in MND according to current literature. The table displays a list of studies with at least one longitudinal follow-up after the first observation for the neurofilament of interest; the main findings are highlighted in bold characters. Methods for NF quantification and the potential limits of these findings are briefly reported

From: Neurofilaments in motor neuron disorders: towards promising diagnostic and prognostic biomarkers

Neurofilament isoform Study Biomatrix Methods Concentration range of the kit Concentration range of cross-sectional results Longitudinal sample size (n) Genetic carriers (n) Longitudinal data Potential limits
NfL Lu, 2015 [56] serum, plasma and CSF ECL-based assay Linearity tested 1–50,000 pg/mL IQR plasma: 54.4–158.4 pg/mL
IQR serum: 54.5–151 pg/mL
IQR CSF: 4376–11,736 pg/mL
Plasma: 67
Serum: 43
CSF: 24
excluded plasma NfL does not change over time
serum NfL have small ns increase over time: stable NfL levels in blood for 15 mts follow-up
CSF NfL: small increase in fast and slow progressors
1) 3 cohorts testing different biomatrices, not related blood and CSF in the same patient.
2) Small sample for CSF collected longitudinally
Skillback, 2017 [84] CSF In-house method [I method] and ELISA kit Uman Diagnostics [II method] (NF-light ELISA kit,
UmanDiagnostics AB, Umea°, Sweden); normalization of the two methods
I method: detection limit 250 ng/mL II method: detection limit 125 ng/mL (linearity 125–16,000 ng/mL)
III method: detection limit 50 ng/mL; linear correlation with previous methods but resulted in higher levels of NfL (normalization required)
970–3600 ng/mL 69 unknown 67% of MND showed higher CSF NFL concentration at a later stage of disease, those without rising levels had higher NFL at baseline 1) use of two different detection methods, the first less sensitive;
2) no notion of the timeline of longitudinal sampling (after how many months the second sample, in which phase of disease, etc)
Poesen, 2017 [69] CSF pNfH: Biovendor, Brno, Czech Republic; NfL: UmanDiagnostics AB, Umea, Sweden; UD51001 pNFH: 62.5–4000 pg/ml NfL: 1–10,000 pg/mL pNFH: 114–18,089 pg/mL NfL: 370–108,909 pg/mL 17 26 /220 in cross-sectional cohort not specified in longitudinal cohort CSF pNfH levels rather stable over time, CSF NfL increased over time for a subset of intermediate and fast disease progressors 1) small sample for longitudinal observation;
2) sampling at different disease duration
Benatar, 2018 [85] serum ECL-based assay reference to previous work [50]
15.6–10,000 pg/mL
21–555 pg/mL 11 3 stable levels of NfL in sporadic ALS 1) small sample size;
2) follow-up very sparse without knowledge of clinical progression
Verde, 2019 [68] serum SiMOA, time-normalized follow-up NfL levels 0.686–500 pg /mL [Quanterix] 14.6–908 pg / mL 29 8 / 224 in cross-sectional cohort Overall stable: the variations in NfL from first to second sample ranged from −69.3 to + 189 pg/mL (median, −0.1 pg/mL). small sample size
Gille, 2019 [71] serum ECL-based assay reference to previous work [50] 15.6–10,000 pg/ml 0.3–1141 pg/mL 16 15 / 149 in cross-sectional cohort relatively stable over time, although an increase could be observed within the first 20 months after onset of disease, irrespective of the disease progression rate 1) small sample for longitudinal observation;
2) sampling at different disease duration;
3) no information of pNfH levels related to clinical outcome
Benatar, 2020 [86] serum SiMOA 0.686–500 pg /mL [Quanterix] 2–369 pg/mL 106 8 relatively stable over time, computation of longitudinal trajectories by average slope: 0.011 log units/ month (95% CI, −0.054 to 0.076) well-conducted prospective longitudinal study representative of ALS population; the only potential limit might be sample collection was at some latency from diagnosis (average: 0.7 year), and the study population was skewed towards a slow progression (ALSFRS-r slope: 0.65/month) than general ALS population (1/month, PRO-ACT)
pNfH Lu, 2015 [82] plasma in-house ELISA for hyperphosphorylated NfH (NfHSMI34) and variably phosphorylated NfH (NfHSMI35) linearity tested for serial dilutions with urea pre-analytical tretament to overcome the “hook effect”, tested in normalized optical density (NOD) not reported 74 7 / 136 in cross-sectional cohort slight increase in plasma pNfH for slow progressors (32), no change for intermediate progressors (24), slight decrease for fast progressors (32) in-house immunoassay, now better technologies for pNfH quantification
McCombe, 2015 [87] serum ELISA kit EnCor Biotechnology Inc., Gainesville, FL, USA linearity not tested; from the manufacturer, linearity for standard curve: 0.156–10 ng/mL Not reported 98 not reported overall rise and then fall in pNfH levels for ALS pts. with at least 3 longitudinal samples; different trajectories according to survival potentially better immunodetection techniques
Gendron, 2017 [88] CSF Meso Scale Discovery immunoassay with mouse antihuman pNFH anti-body and a sulfo-tagged polyclonal anti-pNFH antibody as capture and detection antibodies not reported 50–3119 pg/mL non c9-ALS or non c9-ALS-FTD 117–4671 pg/mL for c9-ALS or c9-ALS-FTD 44 27 no change in longitudinally collected pNfH in c9ALS, c9ALS-FTD (both groups: 27 pts) and non-c9 ALS (17 pts); no difference between fast and slow progressors 1) small sample size;
2) no information of pNfH levels related to clinical outcome
Benatar, 2019 [89] serum CE marked ELISA (Euroimmun AG, Lubeck, Germany) 9.4–1000 pg/mL 639–11,418 pg/mL 16 10 stable levels of serum NfH in ALS pts.; among mutation carriers who convert there is an increase in serum pNfH in advance of the appearance
of manifest disease
1) small sample for longitudinal observation;
2) sampling at different disease duration;
3) no information of pNfH levels related to clinical outcome
Benatar, 2020 [86] serum SiMOA 0–2000 pg/mL 1–976 pg/mL 106 8 relatively stable over time, computation of longitudinal trajectories by average slope: 0.006 log units/month month (95% CI, −0.063 to 0.084) well-conducted prospective longitudinal study representative of ALS population; the only potential limit might be sample collection was at some latency from diagnosis (average: 0.7 year), and the study population was skewed towards a slow progression (ALSFRS-r slope: 0.65/month) than general ALS population (1/month, PRO-ACT)
  1. Abbreviations: NfL neurofilament light chain, pNfH neurofilament heavy chain, CSF cerebrospinal fluid, ECL elettrochemoluminescence assay, ELISA enzyme -linked immunoadsorbent assay, ns not significant, MND motor neuron disorder, mts months, pts. patients, SiMoA single-molecule array
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