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.
Petzold
Axel
A
Department of Neuroinflammation, UCL Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom. a.petzold@ion.ucl.ac.uk
Altintas
Ayse
A
Andreoni
Laura
L
Bartos
Ales
A
Berthele
Achim
A
Blankenstein
Marinus A
MA
Buee
Luc
L
Castellazzi
Massimiliano
M
Cepok
Sabine
S
Comabella
Manuel
M
Constantinescu
Cris S
CS
Deisenhammer
Florian
F
Deniz
Gunnur
G
Erten
Gaye
G
Espiño
Mercedes
M
Fainardi
Enrico
E
Franciotta
Diego
D
Freedman
Mark S
MS
Giedraitis
Vilmantas
V
Gilhus
Nils Erik
NE
Giovannoni
Gavin
G
Glabinski
Andrzej
A
Grieb
Pawel
P
Hartung
Hans-Peter
HP
Hemmer
Bernhard
B
Herukka
Sanna-Kaisa
SK
Hintzen
Rogier
R
Ingelsson
Martin
M
Jackson
Samuel
S
Jacobsen
Steve
S
Jafari
Naghmeh
N
Jalosinski
Marcin
M
Jarius
Sven
S
Kapaki
Elisabeth
E
Kieseier
Bernd C
BC
Koel-Simmelink
Marleen J A
MJ
Kornhuber
Johannes
J
Kuhle
Jens
J
Kurzepa
Jacek
J
Lalive
Patrice H
PH
Lannfelt
Lars
L
Lehmensiek
Vera
V
Lewczuk
Piotr
P
Livrea
Paolo
P
Marnetto
Fabiana
F
Martino
Davide
D
Menge
Til
T
Norgren
Niklas
N
Papuć
Eva
E
Paraskevas
George P
GP
Pirttilä
Tuula
T
Rajda
Cecília
C
Rejdak
Konrad
K
Ricny
Jan
J
Ripova
Daniela
D
Rosengren
Lars
L
Ruggieri
Maddalena
M
Schraen
Susanna
S
Shaw
Gerry
G
Sindic
Christian
C
Siva
Aksel
A
Stigbrand
Torgny
T
Stonebridge
Iva
I
Topcular
Baris
B
Trojano
Maria
M
Tumani
Hayrettin
H
Twaalfhoven
Harry A M
HA
Vécsei
László
L
Van Pesch
Vincent
V
Vanderstichele
Hugo
H
Vedeler
Christian
C
Verbeek
Marcel M
MM
Villar
Luisa Maria
LM
Weissert
Robert
R
Wildemann
Brigitte
B
Yang
Cui
C
Yao
Karen
K
Teunissen
Charlotte E
CE
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