Neurofilament ELISA validation.
BACKGROUND
Neurofilament proteins (Nf) are highly specific biomarkers for neuronal death and axonal degeneration. As these markers become more widely used, an inter-laboratory validation study is required to identify assay criteria for high quality performance.


METHODS
The UmanDiagnostics NF-light (R)enzyme-linked immunoabsorbent assays (ELISA) for the neurofilament light chain (NfL, 68kDa) was used to test the intra-assay and inter-laboratory coefficient of variation (CV) between 35 laboratories worldwide on 15 cerebrospinal fluid (CSF) samples. Critical factors, such as sample transport and storage, analytical delays, reaction temperature and time, the laboratories' accuracy and preparation of standards were documented and used for the statistical analyses.


RESULTS
The intra-laboratory CV averaged 3.3% and the inter-laboratory CV 59%. The results from the test laboratories correlated with those from the reference laboratory (R=0.60, p<0.0001). Correcting for critical factors improved the strength of the correlation. Differences in the accuracy of standard preparation were identified as the most critical factor. Correcting for the error introduced by variation in the protein standards improved the correlation to R=0.98, p<0.0001 with an averaged inter-laboratory CV of 14%. The corrected overall inter-rater agreement was subtantial (0.6) according to Fleiss' multi-rater kappa and Gwet's AC1 statistics.


CONCLUSIONS
This multi-center validation study identified the lack of preparation of accurate and consistent protein standards as the main reason for a poor inter-laboratory CV. This issue is also relevant to other protein biomarkers based on this type of assay and will need to be solved in order to achieve an acceptable level of analytical accuracy. The raw data of this study is available online.
Department of Neuroinflammation, UCL Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom. a.petzold@ion.ucl.ac.uk
Axel
A
Petzold
Ayse
A
Altintas
Laura
L
Andreoni
Ales
A
Bartos
Achim
A
Berthele
Marinus A
MA
Blankenstein
Luc
L
Buee
Massimiliano
M
Castellazzi
Sabine
S
Cepok
Manuel
M
Comabella
Cris S
CS
Constantinescu
Florian
F
Deisenhammer
Gunnur
G
Deniz
Gaye
G
Erten
Mercedes
M
Espiño
Enrico
E
Fainardi
Diego
D
Franciotta
Mark S
MS
Freedman
Vilmantas
V
Giedraitis
Nils Erik
NE
Gilhus
Gavin
G
Giovannoni
Andrzej
A
Glabinski
Pawel
P
Grieb
Hans-Peter
HP
Hartung
Bernhard
B
Hemmer
Sanna-Kaisa
SK
Herukka
Rogier
R
Hintzen
Martin
M
Ingelsson
Samuel
S
Jackson
Steve
S
Jacobsen
Naghmeh
N
Jafari
Marcin
M
Jalosinski
Sven
S
Jarius
Elisabeth
E
Kapaki
Bernd C
BC
Kieseier
Marleen J A
MJ
Koel-Simmelink
Johannes
J
Kornhuber
Jens
J
Kuhle
Jacek
J
Kurzepa
Patrice H
PH
Lalive
Lars
L
Lannfelt
Vera
V
Lehmensiek
Piotr
P
Lewczuk
Paolo
P
Livrea
Fabiana
F
Marnetto
Davide
D
Martino
Til
T
Menge
Niklas
N
Norgren
Eva
E
Papuć
George P
GP
Paraskevas
Tuula
T
Pirttilä
Cecília
C
Rajda
Konrad
K
Rejdak
Jan
J
Ricny
Daniela
D
Ripova
Lars
L
Rosengren
Maddalena
M
Ruggieri
Susanna
S
Schraen
Gerry
G
Shaw
Christian
C
Sindic
Aksel
A
Siva
Torgny
T
Stigbrand
Iva
I
Stonebridge
Baris
B
Topcular
Maria
M
Trojano
Hayrettin
H
Tumani
Harry A M
HA
Twaalfhoven
László
L
Vécsei
Vincent
V
Van Pesch
Hugo
H
Vanderstichele
Christian
C
Vedeler
Marcel M
MM
Verbeek
Luisa Maria
LM
Villar
Robert
R
Weissert
Brigitte
B
Wildemann
Cui
C
Yang
Karen
K
Yao
Charlotte E
CE
Teunissen
D015415:Biomarkers / Q000134:cerebrospinal fluid*; D016923:Cell Death; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D016900:Neurofilament Proteins / Q000134:cerebrospinal fluid*; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D015588:Observer Variation; D012015:Reference Standards; D015203:Reproducibility of Results; D013048:Specimen Handling; D013696:Temperature; D013997:Time Factors
D015415:Biomarkers; D016900:Neurofilament Proteins; C066858:neurofilament protein NF 68