Change in CSF marker classification according to cut-offs.

<p>Results are CSF levels only for subjects in which reanalysis let to a different biomarker classification using the cut-offs from Amsterdam to define abnormal CSF values. On the left (A) are changes in biomarker levels for intralaboratory reanalysis and on the right (B) for interlaboratory reanalysis. The arrow represents the applied Amsterdam CSF cut-off. Analysis 1 is routine practice and analysis 2 is performed as part of the LeARN study. CSF = cerebrospinal fluid, Aβ = amyloid beta, t-tau = total tau, p-tau = phosphorylated tau.</p

Intralaboratory lot-to-lot variation.

<p>Results in this graph are based on deming regression and show the CSF levels of lot 1 and lot 3 for Aβ1-42 (A), t-tau (B), and p-tau (C). The slope is different for Aβ1-42 levels between lot 1 and lot 3. The mean difference in CSF levels between lot 1 and 3 was significantly different for Aβ1-42 and t-tau. Lot 1 = clinical routine lot, lot 3 = LeARN lot, Aβ = amyloid beta, t-tau = total tau, p-tau = phosphorylated tau.</p

Internal control data of the Amsterdam laboratory.

<p>Results are changes in high and low CSF biomarker levels for intralaboratory reanalysis in two lots (routine lot and LeARN lot) as part of internal control of data at the Amsterdam laboratory. A) Aβ1-42: Lot 1 used from February 2010 to February 2011 (n = 18) and lot 2 used from March to October 2012 (n = 17). B) T-tau: Lot 1 used from February 2010 to April 2011 (n = 24) and lot 2 used from March to June 2012 (n = 11). C) P-tau: Lot 1 used from February 2010 to April 2011 (n = 23) and lot 2 used from March to October 2012 (n = 11). CSF = cerebrospinal fluid, Aβ = amyloid beta, t-tau = total tau, p-tau = phosphorylated tau. *P<0.001 for differences between lot of analysis 1 (routine lot) and lot of analysis 2 (LeARN lot).</p

Bland-Altman plots of variability between CSF analyses.

<p>The average of CSF analysis 1 and 2 is plotted against the difference between both analyses, on the left (A) for CSF intralaboratory analyses and on the right (B) for CSF interlaboratory analyses. The solid line represents the mean and the dotted lines the upper and lower 1.95 SD. CSF = cerebrospinal fluid, Aβ = amyloid beta, t-tau = total tau, p-tau = phosphorylated tau.</p

Agreement between CSF analyses.

<p>Results are Pearson correlation and ICC (95% CI) for intra- and interlaboratory CSF analyses. ICC = Intraclass coefficients, ratio = Aβ1-42/t-tau, CSF = cerebrospinal fluid, Aβ = amyloid beta, t-tau = total tau, p-tau = phosphorylated tau.</p><p>*All p<0.001.</p

The relationship between serum biomarkers NfH, NOx, GFAP, BDNF, NGF and clinical (MSFC, EDSS, MSIS-29) as well as MRI measures are presented in tabular form and as forest plots.

<p>a) 25-foot walk (secs); b) 9-hole peg test, dominant had (secs); c) 9-hole peg test, non-dominant hand (secs); d) Paced Auditory Serial Addition Test (PASAT); e) Z-score; f) Expanded Disability Status Scale (EDSS); g) Multiple Sclerosis Impact Scale (MSIS-29) physical; h) MSIS-29 psychological; i) Magnetization Transfer Ratio (MTR) white matter; j) MTR grey matter; k) MTR whole brain; l) MRI Central Cerebral Volume (MRICCV); m) T1 volume; n) T2 volume.</p

Survival curves of walking times and 9-hole peg test times (dominant and non-dominant hand) are plotted according to absent, below median or above median NfH levels.

<p>Survival curves of walking times and 9-hole peg test times (dominant and non-dominant hand) are plotted according to absent, below median or above median NfH levels.</p

Summary characteristics of subjects in the study according to randomisation, serum adherence and tablet compliance (p>0.05).

<p>Three samples were present in each sub-group corresponding to time points 0–12–24 months *, and of the 120 subjects who participated in the biomarker study there were 19 drop-outs ^†.</p

CSF levels by analysis and marker.

<p>Results are frequencies and mean (SD) for each CSF marker on the left (A) for CSF intralaboratory analyses and on the right (B) for CSF interlaboratory analyses. The solid line represents the mean CSF levels. Analysis 1 is routine practice and analysis 2 is performed as part of the LeARN study. CSF = cerebrospinal fluid, Aβ = amyloid beta, t-tau = total tau, p-tau = phosphorylated tau. **P<0.001, *p<0.05 compared to CSF analysis 1 or lab 1.</p

Power curves for determining sample size for changes percentage change in NfH levels.

<p>Power curves for determining sample size for changes percentage change in NfH levels.</p