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

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)