(0.48ng/ml). At RT there was about a 5% decrease (p < 0.05).

Long-term storage (6.5 years) of native CSF at �80�C did not

lead to a significant decrease of NfH from a baseline concentra-

tion of 8ng/ml (see Fig, right part). In line with the ELISA data

the NfHSMI35 band remained stable in the immunoblot.

Taken together the quantitative and qualitative data

shows that NfL is not stable in native CSF. Because quantifica-

tion of NfH from the same samples did not shown a significant

decrease this problem is specific to NfL, possibly related to pro-

tease activity (reviewed in Petzold2).

In order to recommend CSF NfL as a biomarker to be

used in clinical trials and for treatment decisions we would like

to ask the authors to share their own experience on the stability

of NfL. If our concern were to be correct, what are the authors’

recommendations to avoid preanalytical problems?

Acknowledgments

EDAR and cNEUPRO are acknowledged for support of

M.J.K. EDAR is the acronym for beta amyloid oligomers

in the Early Diagnosis of AD and as marker for treat-

ment Response and cNeupro (clinical NEUroPROteo-

mics of neurodegenerative diseases) is a Specific Targeted

Research Project (STREP) within the sixth framework

program of the European Commission dedicated to the

search for novel biomarker candidates for Alzheimer’s

disease and other neurodegenerative diseases.

1Department of Clinical Chemistry, Vrije Universiteit (VU)University Medical Center, Amsterdam, The Netherlands,2Medisch (MS) Centrum Amsterdam, VU University MedicalCenter, Amsterdam, The Netherlands, and 3University CollegeLondon (UCL) Institute of Neurology, Department ofNeuroimmunology, UCL, London, UK

References

1. Gunnarsson M, Malmestrom C, Axelsson M, et al. Axonal damagein relapsing multiple sclerosis is markedly reduced by natalizumab.Ann Neurol 2011;69:83–89.

2. Petzold A. Neurofilament phosphoforms: surrogate markers foraxonal injury, degeneration and loss. J Neurol Sci 2005;233:183–198.

3. Teunissen CE, Dijkstra C, Polman C. Biological markers in CSFand blood for axonal degeneration in multiple sclerosis. LancetNeurol 2005;4:32–41.

4. Teunissen CE, Petzold A, Bennett JL, et al. A consensus protocolfor the standardization of cerebrospinal fluid collection andbiobanking. Neurology 2009;73:1914–1922.

5. Petzold A, Keir G, Green AJE, et al. A specific ELISA formeasuring neurofilament heavy chain phosphoforms. J ImmunolMethods 2003;278:179–190.

DOI: 10.1002/ana.22438

ReplyMartin Gunnarsson, MD, PhD,1 Clas Malmestrom, MD, PhD,2

Lars Rosengren, MD, PhD,2 Jan Lycke, MD, PhD,2

and Anders Svenningsson, MD, PhD3

Koel-Simmelink and colleagues raise issues regarding the

reliability of neurofilament light protein (NFL) as a bio-

marker in clinical trials and to facilitate treatment decisions

in multiple sclerosis (MS). The authors focus on preanalyti-

cal sample handling in terms of susceptibility for NFL deg-

radation. Koel-Simmelink et al. present data suggesting that

NFL levels in cerebrospinal fluid (CSF) are significantly

decreased following storage at 4�C or room temperature

(RT) overnight. In another study by Van Geel and

colleagues,1 no decrease in NFL concentration was obtained

following storage for 3 days at 4�C. We performed analyses

on a set of CSF samples with different storage conditions

and found on average 7.1% decrease of NFL concentration

after 24 hours storage at 4�C and 13% decrease at RT

FIGURE: A set of CSF samples (n 2 9) at a wideconcentration range of NFL were analyzed immediatelyafter lumbar puncture and after storage at 4�C at differenttime points or at RT. (A) The mean decay of NFLconcentration after 24 hours storage at 4�C was 7.1% andat RT 13%. Using 1-sided paired t test the differences wereborderline significant (p 5 0.045–0.052). (B) Stability wasequally good over the whole range of NFL concentrations.NFL 5 neurofilament light protein; CSF 5 cerebrospinalfluid; RT 5 room temperature.

1066 Volume 69, No. 6

ANNALS of Neurology

(Fig). Different methods for NFL detection may explain

such divergent results. An extensive amount of previous data

has shown that phosphorylated neurofilament heavy protein

(pNFH) is more stable against proteolysis as compared to

NFL.2 On the other hand, pNFH is susceptible to dephos-

phorylation, whereas NFL is not. A changed degree of

phosphorylation of NFH has been implied in several neuro-

logical disorders2 and in this aspect the assay of CSF NFL

is advantageous. Our earlier studies and the present work

using NFL as biomarker circumvent this potential problem.

CSF NFL levels reflect disease activity in the early phase of

MS; ie, levels in CSF are measurable in remission and sig-

nificantly increased during exacerbations. Moreover, the

UmanDiagnostics NF-lightVR

enzyme-linked immunosorbent

assay (ELISA) is highly sensitive, also enabling quantification

of NFL in healthy subjects. By taking the issue of stability

among other factors into consideration, we agree with

Koel-Simmelink and colleagues that accurate sample

handling is a crucial step in CSF biomarker analysis.

Thus, all samples in our study, before and after 6 to 12

months of natalizumab treatment, were snap-frozen to �80�Cwithin 6 hours of sampling and not thawed until analysis.3

This is in line with the recently published consensus protocol

for standardization of CSF sample collection.4 In such condi-

tions, no problems with NFL degradation can be anticipated.

The multicenter design of our study also supports the feasibility

of this preanalytical sample handling. NFL levels in the CSF

were markedly reduced following natalizumab treatment and

posttreatment levels were not significantly different from those

obtained in healthy subjects.3 Our results suggest that NFL has

the potential to provide treatment efficacy information. Further

investigation on pNFH in this CSF sample set and others are

warranted. Such studies are in progress.

Potential Conflicts of Interest

Nothing to report.

1Department of Neurology, Orebro University Hospital, Orebro,2Department of Neurology, Sahlgrenska University Hospital,Goteborg, and 3Department of Pharmacology and ClinicalNeuroscience, Umea University, Umea, Sweden

References

1. Van Geel WJA, Rosengren LE, Verbeek MM. An enzyme immuno-assay to quantify neurofilament light chain in cerebrospinal fluid. JImmunol Methods 2005;296:179–185.

2. Petzold A. Neurofilament phosphoforms: surrogate markers for axonalinjury, degeneration and loss. J Neurol Sci 2005;233:183–198.

3. Gunnarsson M, Malmestrom C, Axelsson M, et al. Axonal damagein relapsing multiple sclerosis is markedly reduced by natalizumab.Ann Neurol 2011;69:83–88.

4. Teunissen CE, Petzold A, Bennett JL, et al. A consensus protocolfor the standardization of cerebrospinal fluid collection and bio-banking. Neurology 2009;73:1914–1922.

DOI: 10.1002/ana.22450

Vitamin B12, Folate and Hyperhomocysteinemia inPatients with EpilepsyVincenzo Belcastro, MD,1 and Pasquale Striano, MD, PhD2

We read with great interest the study by Linnebank and

coworkers1 showing that some of the old (eg, carbamazepine,

phenobarbital, phenytoin, primidone) and new (eg, oxcarbaze-

pine, topiramate, pregabalin) antiepileptic drugs (AEDs) inter-

act with folate and vitamin B12 serum levels and in turn raise

homocysteine (Hcy) levels in patients with epilepsy.

Epidemiologic studies have suggested that patients with

epilepsy have a greater prevalence of cardiovascular and cerebro-

vascular disease than normally observed in the general popula-

tion2 and, among the various variables analyzed, hyperhomocys-

teinemia (hyper-Hcy) has been indicated as an independent risk

factor for the above mentioned conditions.3 In this sense, the

implications of the work by Linnebank and coworkers1 are con-

siderable, as (1) it is the largest sample size study investigating

the chronic effect of AEDs on Hcy serum levels and (2) it poses

the challenging question as to whether to use inducing anticon-

vulsants as first-line agents for long-term treatment of epilepsy.

Since a condition of hyper-Hcy would be easily corrected by

intake of folate and other B vitamins, Linnebank and col-

leagues1 suggest that prophylactic folate supplementation (eg,

5mg or 1mg) may be considered to decrease Hcy levels and to

reduce side effects mediated by reduced folate levels. Notewor-

thy, hyper-Hcy recurrence is also influenced by the presence of

common polymorphisms of the methylene-tetrahydrofolate-re-

ductase (MTHFR) gene in patients with epilepsy.4

An additional aspect to consider is that chronic folate ther-

apy may exert adverse effects, including a putative epileptogenic

action, a carcinogenic effect, complication of the diagnosis of per-

nicious anemia, and seizure precipitation due to a lowering effect

on plasma AED levels.5 For these reasons, although hyper-Hcy

has been indicated to be an independent risk factor for atheroscle-

rosis in epilepsy patients,3 the question remains unanswered as to

whether or not hyper-tHcy (>15 lm) is a mere biochemical

marker of atherogenesis or a clear causative factor.6

Although a clear association between elevated plasma Hcy

levels and vascular disease/brain atrophy has not been definitely

demonstrated, we agree with Linnebank and colleagues1 that

the knowledge of laboratory changes (eg, folate, vitamin B12,

Hcy serum levels) as well as of the screening for MTHFR gene

polymorphisms may be helpful in optimizing the AED therapy

by folate supplementation pulses in patients with epilepsy.

1Neurology Clinic, Department of Neuroscience, Sant’AnnaHospital, Como, Italy 2Muscular and NeurodegenerativeDiseases Unit, G. Gaslini Institute, University of Genova, Italy

References

1. Linnebank M, Moskau S, Semmler A, et al. Antiepileptic drugsinteract with folate and vitamin B12 serum levels. Ann Neurol2011;69:352–359.

2. Annegers JF, Hauser WA, Shirts SB. Heart disease mortalityand morbidity in patients with epilepsy. Epilepsia 1984;25:699–704.

June 2011 1067