Pre-analytical stability of novel cerebrospinal fluid biomarkers.
Author(s): Willemse EAJ, Vermeiren Y, Garcia-Ayllon MS, Bridel C, De Deyn PP, Engelborghs S, van der Flier WM, Jansen EEW, Lopez-Font IB, Mendes V, Manadas B, de Roeck N, Saez-Valero J, Struys EA, Vanmechelen E, Andreasson U, Teunissen CE
Publication: Clin Chim Acta, 2019, Vol. 497, Page 204-211
PubMed ID: 31348908 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of short-term storage at various temperatures and freeze-thaw cycling of cerebrospinal fluid (CSF) on biomarkers used for the diagnosis of neurological diseases.
Conclusion of Paper
Levels of 3-Methoxy-4-hydroxyphenylglycol (MHPG) decreased significantly after more than 4 h at room temperature, 24 h at 4°C, or 4 months at -20°C and decreased by 6.4% after every freeze/thaw cycle. Levels of 5-Methyltetrahydrofolate (5-MTHF) and 5-hydroxyindoleacetic acid levels (5-HIAA) declined after 1 week at room temperature. Levels of 3,4-dihydroxyphenylacetic acid (DOPAC) increased after >24 h at room temperature or 4°C and after one week at -20°C. Levels of neurogranin, chitinase-3-like protein (YKL-40), beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), acetylcholinesterase (AChE) enzymatic activity, theobromine, secreted protein acidic and rich in cysteine-like 1 (SPARCL-1), and homovanillic acid (HVA) were not affected by delayed storage duration at any of the timepoints examined or by freeze/thaw cycling up to 7 times.
Studies
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Study Purpose
This study investigated the effects of short-term storage at various temperatures and freeze-thaw cycling of cerebrospinal fluid (CSF) on biomarkers used for the diagnosis of neurological diseases. Surplus CSF specimens stored at −80°C in the Alzheimer Center Biobank at the Amsterdam University Medical Center were pooled from 3-6 individuals and then aliquoted into polypropylene tubes. Tubes were stored for 0, 1, 2, 4, 24, 72, or 168 h at 4°C or room temperature; for 1–4 months at −20°C; or subjected to up to 7 freeze/thaw (f/t) cycles before final storage at −80°C for up to one year. Neurogranin, YKL-40, SPARCL-1, BACE1 levels were measured by enzyme-linked immunosorbent assay (ELISA). To investigate the effects of delayed storage on 5-MTHF (a metabolite of folic acid), fresh CSF was centrifuged at 2000 x g for 10 min and then stored for 30 min, 1 h, 1 day, or 1 week at 4 °C or RT or subjected to 1-4 freeze/thaw cycles before storage at −80 °C for up to one year. Theobromine levels were measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS). AChE activity was determined by a modified microassay version of the colorimetric Ellman's method. Metabolites of noradrenaline, dopamine, and serotonin (MHPG, DOPAC and HVA, and 5-HIAA; respectively) and 5-MTHF were measured using a reversed phase ultra-high pressure liquid chromatography system coupled with electrochemical detection (RP-UHPLCECD).
Summary of Findings:
Levels of 5-HIAA declined after 1 week at 4°C, 3 days at room temperature, or 2 months at −20°C, but were not affected by freeze/thaw cycling up to 7 times. In contrast, MHPG levels steadily decreased after 4 h at 4°C or room temperature 1 month at −20°C and decreased by 6.4% after every freeze/thaw cycle. DOPAC levels slightly increased after 24 h storage at 4°C or room temperature and after 2 months at −20°C but were unaffected by 7 freeze/thaw cycles. 5-MTHF levels were not affected by 4 freeze/thaw cycles but decreased sharply after 1 week of storage at 4°C and declined by 30% after 1 week at room temperature. Levels of neurogranin, YKL-40, theobromine, SPARCL-1, BACE1, AChE, and HVA were not affected by delayed storage of over 1 week at 4 °C or room temperature or 1 month at −20°C or by freeze/thaw cycling up to 7 times.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Alzheimer's Disease
Platform:
Analyte Technology Platform Small molecule ELISA Small molecule HPLC Small molecule Colorimetric assay Small molecule LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 h
1 h
2 h
4 h
24 h
72 h
168 h
Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
3 cycles
4 cycles
5 cycles
6 cycles
7 cycles
Storage Storage temperature Room temperature
4°C
−20°C