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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

Conclusion of Paper

Studies

1. 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

   • Bodily Fluid - Cerebrospinal Fluid

   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

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