Analytical and pre-analytical aspects of neurofilament light chain determination in biological fluids

Authors: L. Fialová 1;  L. Nosková 1;  M. Kalousová 1;  T. Zima 1;  T. Uher 2;  A. Bartoš 3
Authors‘ workplace: Ústav lékařské biochemie a laboratorní, diagnostiky, 1. LF UK a VFN v Praze 1;  Centrum pro demyelinizační onemocnění, Neurologická klinika, 1. LF UK a VFN v Praze 2;  Neurologická klinika 3. LF UK, Praha 3
Published in: Cesk Slov Neurol N 2022; 85(1): 11-16
Category: Review Article
doi: 10.48095/cccsnn202211


The aim of this review is to inform clinical and laboratory workers about the most important pre-analytical and analytical aspects of neurofilament light chain (NfL) determination in bio­logical fluids. NfLs represent a promising nonspecific bio­marker of neuronal and axonal damage that occurs in a variety of neurological diseases. Before introducing NfL determination into routine clinical practice, it is necessary to characterize the pre-analytical and analytical aspects of the assays, which can significantly affect the accuracy of the analysis results. When evaluating NfL concentrations, the patient‘s age should be taken into account and body mass index may also have an effect. The advantages of NfLs are their long-term storage stability at different temperatures as well as resistance to repeated freezing and thawing cycles. NfL concentrations in clinical trials are determined primarily by immunoassay methods that vary in sensitivity. There are several immunoassay technologies for the determination of NfL suitable for reliable determination in cerebrospinal fluid (CSF) and serum/plasma. The choice of the optimal analytical approach depends, among other things, on the concentration of NfL in bio­logical fluids. ELISA methods can be used to determine NfL in CSF, which show sufficient sensitivity for higher concentrations of NfL occurring in this bio­logical fluid. Newly introduced technologies characterized by significantly higher sensitivity in comparison with the ELISA methods enabled reliable examination of NfL also in serum/plasma. The principles of methods based on Simoa® technology, SimplePlexTM, and immunomagnetic reduction are mentioned in more detail.


Blood – cerebrospinal fluid – biomarker – neurofilaments – pre-analytical phase


1. Thebault S, Booth RA, Freedman MS. Blood neuro­filament light chain: the neurologist‘s troponin? Biomedicines 2020; 8 (11): 523. doi: 10.3390/bio­medicines8110 523.

2. Yuan A, Rao MV, Veeranna et al. Neurofilaments and neurofilament proteins in health and disease. Cold Spring Harb Perspect Biol 2017; 9 (4): a018309. doi: 10.1101/cshperspect.a018309.

3. Lobsiger CS, Cleveland DW. Neurofilaments: organization and function in neurons. In: Squire LR (eds). Encyclopedia of neuroscience. Amsterdam: Elsevier 2009: 433–436. doi: 10.1016/B978-008045046-9.00728-2.

4. Bridel C, van Wieringen WN, Zetterberg H et al. Dia­g­- nostic value of cerebrospinal fluid neurofilament light protein in neurology: a systematic review and meta-analysis. JAMA Neurol 2019; 76 (9): 1035–1048. doi: 10.1001/jamaneurol.2019.1534.

5. Disanto G, Barro C, Benkert P et al. Serum neurofilament light: a bio­marker of neuronal damage in multiple sclerosis. Ann Neurol 2017; 81 (6): 857–870. doi: 10.1002/ana.24954.

6. Fialová L. Neurofilamenta u traumatického poškození mozku – současné znalosti. Klin Biochem Metab 2018; 26 (2): 68–75.

7. Piťha J. Biomarkery roztroušené sklerózy – současné možnosti a perspektivy. Cesk Slov Neurol N 2015; 78/111 (3): 269–273.

8. Kusnierova P, Zeman D, Hradilek P et al. Neurofilament levels in patients with neurological diseases: a comparison of neurofilament light and heavy chain levels. J Clin Lab Anal 2019; 33 (7): e22948. doi: 10.1002/jcla.22948.

9. Khalil M, Teunissen CE, Otto M et al. Neurofilaments as bio­markers in neurological disorders. Nat Rev Neurol 2018; 14 (10): 577–589. doi: 10.1038/s41582-018-0058-z.

10. Gaetani L, Blennow K, Calabresi P et al. Neurofilament light chain as a bio­marker in neurological disorders. J Neurol Neurosurg Psychiatry 2019; 90 (8): 870–881. doi: 10.1136/jnnp-2018-320106.

11. Andersson E, Janelidze S, Lampinen B et al. Blood and cerebrospinal fluid neurofilament light differentially detect neurodegeneration in early Alzheimer‘s disease. Neurobio­l Aging 2020; 95: 143–153. doi: 10.1016/j.neurobio­laging.2020.07.018.

12. Rosengren LE, Karlsson JE, Karlsson JO et al. Patients with amyotrophic lateral sclerosis and other neurodegenerative diseases have increased levels of neurofilament protein in CSF. J Neurochem 1996; 67 (5): 2013–2018. doi: 10.1046/j.1471-4159.1996.67052013.x.

13. Kuhle J, Barro C, Andreasson U et al. Comparison of three analytical platforms for quantification of the neurofilament light chain in blood samples: ELISA, electrochemiluminescence immunoassay and Simoa. Clin Chem Lab Med 2016; 54 (10): 1655–1661. doi: 10.1515/cclm-2015-1195.

14. Fialova L, Bartos A, Svarcova J et al. Serum and cerebrospinal fluid light neurofilaments and antibodies against them in clinically isolated syndrome and multiple sclerosis. J Neuroimmunol 2013; 262 (1–2): 113–120. doi: 10.1016/j.jneuroim.2013.06.010.

15. Srpova B, Uher T, Hrnciarova T et al. Serum neurofilament light chain reflects inflammation-driven neurodegeneration and predicts delayed brain volume loss in early stage of multiple sclerosis. Mult Scler 2021; 27 (1): 52–60. doi: 10.1177/1352458519901272.

16. Fialova L. Neurofilaments in multiple sclerosis. Multiple Sclerosis News 2019; 5 (1): 6–12.

17. Uher T, Schaedelin S, Srpova B et al. Monitoring of radiologic disease activity by serum neurofilaments in MS. Neurol Neuroimmunol Neuroinflamm 2020; 7 (4): e714. doi: 10.1212/nxi.0000000000000714.

18. Uher T, Kubala Havrdova E, Benkert P et al. Measurement of neurofilaments improves stratification of future disease activity in early multiple sclerosis. Mult Scler 2021; 27 (13): 2001–2013. doi: 10.1177/13524585211047977.

19. Barro C, Benkert P, Disanto G et al. Serum neurofilament as a predictor of disease worsening and brain and spinal cord atrophy in multiple sclerosis. Brain 2018; 141 (8): 2382–2391. doi: 10.1093/brain/awy154.

20. Barro C, Chitnis T, Weiner HL. Blood neurofilament light: a critical review of its application to neurologic disease. Ann Clin Transl Neurol 2020; 7 (12): 2508–2523. doi: 10.1002/acn3.51234.

21. Fialova L, Bartos A, Svarcova J. Neurofilaments and tau proteins in cerebrospinal fluid and serum in dementias and neuroinflammation. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2017; 161 (3): 286–295. doi: 10.5507/bp.2017.038.

22. Shahim P, Politis A, van der Merwe A et al. Neurofilament light as a bio­marker in traumatic brain injury. Neurology 2020; 95 (6): e610–e622. doi: 10.1212/wnl.0000000 000009983.

23. Moseby-Knappe M, Mattsson N, Nielsen N et al. Serum neurofilament light chain for prognosis of outcome after cardiac arrest. JAMA Neurol 2019; 76 (1): 64–71. doi: 10.1001/jamaneurol.2018.3223.

24. Alagaratnam J, von Widekind S, De Francesco D et al. Correlation between CSF and blood neurofilament light chain protein: a systematic review and meta-analysis. BMJ Neurol Open 2021; 3 (1): e000143. doi: 10.1136/bmjno-2021-000143.

25. Gleerup HS, Sanna F, Hogh P et al. Saliva neurofilament light chain is not a dia­gnostic bio­marker for neurodegeneration in a mixed memory clinic population. Front Aging Neurosci 2021; 13: 659898. doi: 10.3389/fnagi.2021.659898.

26. Hviid CVB, Knudsen CS, Parkner T. Reference interval and preanalytical properties of serum neurofilament light chain in Scandinavian adults. Scand J Clin Lab Invest 2020; 80 (4): 291–295. doi: 10.1080/00365513.2020.1730434.

27. Khalil M, Pirpamer L, Hofer E et al. Serum neurofilament light levels in normal aging and their association with morphologic brain changes. Nat Commun 2020; 11 (1): 812. doi: 10.1038/s41467-020-14612-6.

28. Mattsson N, Andreasson U, Zetterberg H et al. Association of plasma neurofilament light with neurodegeneration in patients with Alzheimer disease. JAMA Neurol 2017; 74 (5): 557–566. doi: 10.1001/jamaneurol.2016.6 117.

29. Akamine S, Marutani N, Kanayama D et al. Renal function is associated with blood neurofilament light chain level in older adults. Sci Rep 2020; 10 (1): 20350. doi: 10.1038/s41598-020-76990-7.

30. Korley FK, Goldstick J, Mastali M et al. Serum NfL (Neurofilament Light Chain) levels and incident stroke in adults with diabetes mellitus. Stroke 2019; 50 (7): 1669–1675. doi: 10.1161/STROKEAHA.119.024941.

31. Howell JC, Watts KD, Parker MW et al. Race modifies the relationship between cognition and Alzheimer‘s disease cerebrospinal fluid bio­markers. Alzheimers Res Ther 2017; 9 (1): 88. doi: 10.1186/s13195-017-0315-1.

32. Manouchehrinia A, Piehl F, Hillert J et al. Confounding effect of blood volume and body mass index on blood neurofilament light chain levels. Ann Clin Transl Neurol 2020; 7 (1): 139–143. doi: 10.1002/acn3.50972.

33. Wentz E, Dobrescu SR, Dinkler L et al. Thirty years after anorexia nervosa onset, serum neurofilament light chain protein concentration indicates neuronal injury. Eur Child Adolesc Psychiatry 2021; 30 (12): 1907–1915. doi: 10.1007/s00787-020-01657-7.

34. Nilsson IAK, Millischer V, Karrenbauer VD et al. Plasma neurofilament light chain concentration is increased in anorexia nervosa. Transl Psychiatry 2019; 9 (1): 180. doi: 10.1038/s41398-019-0518-2.

35. Hviid CVB, Madsen AT, Winther-Larsen A. Biological variation of serum neurofilament light chain. Clin Chem Lab Med 2021 [ahead of print]. doi: 10.1515/ cclm-2020-1276.

36. Koel-Simmelink MJ, Vennegoor A, Killestein J et al. The impact of pre-analytical variables on the stability of neurofilament proteins in CSF, determined by a novel validated SinglePlex Luminex assay and ELISA. J Immunol Methods 2014; 402 (1–2): 43–49. doi: 10.1016/ j.jim.2013.11.008.

37. Gaiottino J, Norgren N, Dobson R et al. Increased neurofilament light chain blood levels in neurodegenerative neurological diseases. PLoS One 2013; 8 (9): e75091. doi: 10.1371/journal.pone.0075091.

38. Altmann P, Ponleitner M, Rommer PS et al. Seven day pre-analytical stability of serum and plasma neurofilament light chain. Sci Rep 2021; 11 (1): 11034. doi: 10.1038/s41598-021-90639-z.

39. Ashton NJ, Suarez-Calvet M, Karikari TK et al. Effects of pre-analytical procedures on blood bio­markers for Alzheimer‘s pathophysiology, glial activation, and neurodegeneration. Alzheimers Dement (Amst) 2021; 13 (1): e12168. doi: 10.1002/dad2.12168.

40. O‘Connell GC, Alder ML, Webel AR et al. Neuro bio­marker levels measured with high-sensitivity digital ELISA differ between serum and plasma. Bioanalysis 2019; 11 (22): 2087–2094. doi: 10.4155/bio­-2019-0213.

41. Terasawa K, Taguchi T, Momota R et al. Intermediate filaments of endoskeleton within human erythrocytes. Blood 2007; 110 (11): 1734. doi: 10.1182/blood.V110.11.1734.1734.

42. Lombardi V, Carassiti D, Giovannoni G et al. The potential of neurofilaments analysis using dry-blood and plasma spots. Sci Rep 2020; 10 (1): 97. doi: 10.1038/s41598-019-54310-y.

43. Simren J, Ashton NJ, Blennow K et al. Blood neurofilament light in remote settings: alternative protocols to support sample collection in challenging pre-analytical conditions. Alzheimers Dement (Amst) 2021; 13 (1): e12145. doi: 10.1002/dad2.12145.

44. Norgren N, Rosengren L, Stigbrand T. Eleva­ted neurofilament levels in neurological diseases. Brain Res 2003; 987 (1): 25–31. doi: 10.1016/s0006-8993 (03) 03 219-0.

45. Norgren N, Karlsson JE, Rosengren L et al. Monoclonal antibodies selective for low molecular weight neurofilaments. Hybrid Hybridomics 2002; 21 (1): 53–59. doi: 10.1089/15368590252917647.

46. Gauthier A, Viel S, Perret M et al. Comparison of Simoa and Ella to assess serum neurofilament-light chain in multiple sclerosis. Ann Clin Transl Neurol 2021; 8 (5): 1141–1150. doi: 10.1002/acn3.51355.

47. Liu HC, Lin WC, Chiu MJ et al. Development of an assay of plasma neurofilament light chain utilizing immunomagnetic reduction tec hnology. PLoS One 2020; 15 (6): e0234519. doi: 10.1371/journal.pone.0234519.

48. Fialova L, Zima T, Bartos A. Přehled imunoanalytic­kých metod ke stanovení tripletu bio­markerů Alzheimerovy nemoci v mozkomíšním moku a v krvi. Che­mické listy 2020; 114 (8): 537−544.

49. Cohen L, Walt DR. Single-molecule arrays for protein and nucleic acid analysis. Annu Rev Anal Chem (Palo Alto Calif) 2017; 10 (1): 345–363. doi: 10.1146/annurev-anchem-061516-045340.

50. Lue LF, Guerra A, Walker DG. Amyloid beta and tau as Alzheimer’s disease blood bio­markers: promise from new technologies. Neurol Ther 2017; 6 (Suppl 1): 25–36. doi: 10.1007/s40120-017-0074-8.

51. Hendricks R, Baker D, Brumm J et al. Establishment of neurofilament light chain Simoa assay in cerebrospinal fluid and blood. Bioanalysis 2019; 11 (15): 1405–1418. doi: 10.4155/bio­-2019-0163.

52. Korley FK, Yue JK, Wilson DH et al. Performance evaluation of a multiplex assay for simultaneous detection of four clinically relevant traumatic brain injury bio­markers. J Neurotrauma 2018; 36 (1): 182–187. doi: 10.1089/neu.2017.5623.

53. Dysinger M, Marusov G, Fraser S. Quantitative analysis of four protein bio­markers: an automated microfluidic cartridge-based method and its comparison to colorimetric ELISA. J Immunol Methods 2017; 451: 1–10. doi: 10.1016/j.jim.2017.08.009.

54. Yang CC, Yang SY, Chen HH et al. Effect of molecule-particle binding on the reduction in the mixed-frequency alternating current magnetic susceptibility of magnetic bio­-reagents. J Appl Phys 2012; 112 (2): 024704. doi: 10.1063/1.4739735.

55. Plavina T, Rudick RA, Calabresi PA et al. Development of a sensitive serum neurofilament light assay on Siemens routine immunoassay platforms. Multiple Sclerosis J 2019; 25 (2 Suppl): 278. doi: 10.1177/1352458519868 078.

56. Lundberg M, Eriksson A, Tran B et al. Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood. Nucleic Acids Res 2011; 39 (15): e102. doi: 10.1093/nar/gkr424.

57. Šilhán D, Ibrahim I, Tintěra J et al. Parietální atrofie na magnetické rezonanci mozku u Alzheimerovy nemoci s pozdním začátkem. Cesk Slov Neurol N 2019; 82 (1): 91–95. doi: 10.14735/amcsnn201991.

58. Bartoš A, Polanská H. Správná a chybná pojmenování obrázků pro náročnější test písemného Pojmenování obrázků a jejich vybavení (dveřní POBAV). Cesk Slov Neurol N 2021; 84/117 (2): 151–163. doi: 10.48095/cccsnn202115.

59. Bartoš A, Čechová L, Švarcová J et al. Likvorový triplet (tau proteiny a beta-amyloid) v dia­gnostice Alzheimerovy-Fischerovy nemoci. Cesk Slov Neurol N 2012; 75/108 (5): 587–594.

60. Stengelin M, Bathala P, Wohlstadter JN. Sensitive serum/plasma neurofilament light immunoassay. Alzheimers Dement 2019; 15 (7): P1346–P1347. doi: 10.1016/j.jalz.2019.06.3846.

Paediatric neurology Neurosurgery Neurology

Article was published in

Czech and Slovak Neurology and Neurosurgery

Issue 1

2022 Issue 1

Most read in this issue
Forgotten password

Enter the email address that you registered with. We will send you instructions on how to set a new password.


Don‘t have an account?  Create new account