The impact of pre-analytical variables on the stability of neurofilament proteins in CSF, determined by a novel validated SinglePlex Luminex assay and ELISA.
Author(s): Koel-Simmelink MJ, Vennegoor A, Killestein J, Blankenstein MA, Norgren N, Korth C, Teunissen CE
Publication: J Immunol Methods, 2014, Vol. 402, Page 43-9
PubMed ID: 24275679 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of storage temperature and duration on the measurement of neurofilament heavy (NfH) and neurofilament light (NfL) proteins in cerebrospinal fluid (CSF). The effects of processing delays, blood contamination, and freeze-thaw cycling were also investigated using CSF spiked with bovine NfH and NfL.
Conclusion of Paper
NfL concentrations stayed similar to the reference sample concentration (stored at -80°C) after up to 21 days of CSF storage, regardless of storage temperature (-20°C, 4°C, 20°C, or 37°C). However, NfH concentrations declined during storage of CSF at all 4 temperatures, with the largest decreases observed in specimens stored at 37°C (29% of reference sample value after 21 days), and the smallest decreases observed in specimens stored at -20°C (82% of reference sample value after 21 days). Recovery of bovine NfH and NfL was not affected by up to 24 h of delays in CSF processing at either room temperature or 4°C or by up to 4 freeze-thaw cycles. Blood contamination at the 0.5% and 5% levels led to significant increases in measured levels of NfL after room temperature delays and smaller increases after delays at 4°C. However, NfH was unaffected by CSF blood contamination.
Studies
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Study Purpose
The purpose of this study was to determine the effects of storage temperature and duration on the measurement of NfH and NfL proteins in CSF. A single pooled CSF specimen was used for the measurement of NfL stability, while 4 individual CSF specimens were used to determine NfH stability. After experimental storage, all specimens were frozen at -80°C until analysis. NfH and NfL were each measured at a subset of listed timepoints.
Summary of Findings:
NfL concentrations stayed similar to the reference sample concentration (stored at -80°C) after up to 21 days of storage, regardless of storage temperature. However, NfH concentrations declined during storage at all 4 temperatures, with the largest decreases observed in specimens stored at 37°C (29% of reference sample value after 21 days), and the smallest decreases observed in specimens stored at -20°C (82% of reference sample value after 21 days). The authors calculate there is no decrease in NfH concentrations expected after 1 year of CSF storage at -80°C, but they expect 100% loss of NfH after 146 days of -20°C storage.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform Protein Immunoassay Protein ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 d
1 d
2 d
3 d
5 d
6 d
7 d
8 d
9 d
12 d
13 d
14 d
15 d
16 d
17 d
19 d
21 d
Storage Storage temperature -80°C
-20°C
4°C
20°C
37°C
-
Study Purpose
The purpose of this study was to determine the effects of delayed processing, freeze-thaw cycling, and blood contamination on the measurement of bovine NfH and NfL spiked into human CSF. 4 specimens were used in the delayed processing and blood contamination experiments while 14 specimens were used for the freeze-thaw cycling experiments. Freeze-thaw cycling included a 2 h thaw at room temperature with freezing at -80°C for a minimum of 24 h.
Summary of Findings:
Recovery of bovine NfH and NfL was not affected by up to 24 h of delays in CSF processing at either room temperature or 4°C. Blood contamination at the 0.5% and 5% levels led to significant increases in measured levels of NfL after room temperature delays and smaller increases after delays at 4°C. However, NfH was unaffected by CSF blood contamination. Up to 4 freeze-thaw cycles did not result in any consistent or significant effects on the measurement of bovine NfH or NfL in spiked human CSF.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform Protein Immunoassay Protein ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature Room temperature
4°C
Storage Freeze/thaw cycling 1 cycle
2 cycles
4 cycles
Biospecimen Aliquots and Components Biospecimen components 0.625-20 ng/mL bovine NfH
0.625-20 ng/mL bovine NfL
0.05% blood contamination
0.5% blood contamination
5% blood contamination
Storage Storage duration 1 h
2 h
4 h
6 h
24 h