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Preanalytical Stability of CSF Total and Oligomeric Alpha-Synuclein.

Author(s): Abdi IY, Majbour NK, Willemse EAJ, van de Berg WDJ, Mollenhauer B, Teunissen CE, El-Agnaf OM

Publication: Front Aging Neurosci, 2021, Vol. 13, Page 638718

PubMed ID: 33762924 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper compared levels of total and oligomeric alpha-synuclein (α-syn) in cerebrospinal fluid (CSF) specimens that (i) were stored in six different tube types at -80°C, (ii) experienced up to six tube transfers, (iii) were exposed to three different detergents, (iv) were subjected to multiple freeze-thaw cycles, and (v) stored for 1-72 h at room temperature or 4°C.  An unspecified number of anonymous CSF specimens from a biobank stored at -80°C in Sarstedt 1.5 ml polypropylene tubes for an unspecified amount of time were thawed on ice, pooled (referred to as the main sample stock), and aliquoted (500 µl) into six tubes (0.6 ml siliconized Nunc tube (reference), 15 ml Corning tube, 0.5 ml Sarstedt tube, 1.5 ml Eppendorf safe Lock tube, 1.5 ml Extragene tube, 2 ml Costar tube) and stored at -80°C overnight before analysis the following day.  All other experiments used Sarstedt 0.5 ml polypropylene tubes. CSF aliquots of the main sample stock were also analyzed without detergent or after exposure to one of four non-ionic detergents (0.5% Tween-20, 0.5% Triton X-100, 0.5% NP-40) and incubation for 30 min at 4°C. All thawed CSF samples were also transferred to new tubes Sarstedt tubes (0.5 ml polypropylene tubes) 1, 2, 3, 4, 5, or 6 times, with sample mixing after each sample transfer. Samples from the CSF mail sample stock were aliquoted into Sarstedt (0.5 ml) polypropylene tubes and subjected to 1-7 freeze-thaw cycles (no details on the temperature or duration of thaw were provided).  Another set of aliquots from the CSF main sample stock were stored for 1, 2, 4, 24, 72, and 168 h at either room temperature or 4°C before being transferred to -80°C until analysis. Total and oligomeric α-syn protein levels were quantified by an in-house sandwich-based ELISA; using the 11D12 monoclonal antibody (1:5000 dilution) against total α-syn and the FL-140 polyclonal (1:1000 dilution) against oligomeric α-syn.

   Summary of Findings:

   Relative to the reference tube (Nunc tube), reductions in the protein concentration of both total and oligomeric α-syn were observed among the tube types examined after overnight storage at -80°C. Total α-syn concentration was also significantly reduced in CSF samples stored overnight at -80°C in Sarstedt tubes (reduced by 44%, p<0.001), Eppendorf safe Lock 1.5 ml tubes (reduced by 23%, p<0.05), and Extragene 1.5 ml tubes (reduced by 35%, p<0.05) relative to concentrations in CSF samples stored in reference Nunc tubes; reductions that were observed in the remaining tube types were not significant.  Oligomeric α-syn concentration was significantly reduced in CSF samples stored overnight at -80°C in Eppendorf safe Lock 1.5 ml tubes (reduced by 34%, P<0.01) and Extragene 1.5 ml tubes (reduced by 51%, P<0.05) relative to concentrations in CSF samples stored in reference Nunc tubes; reductions that were observed in the remaining tube types were not significant.  CSF samples stored in siliconized tubes (Denville Scientific) demonstrated the smallest reduction in both total and oligomeric α-syn concentration relative to the reference tube (13.5% and 19.5%, respectively).

   The inclusion of the non-ionic detergent 0.5% Tween-20 after thaw and before analysis resulted in a significant 49.2% increase in oligomeric α-syn concentration compared to samples with no detergent (p=0.046); a nonsignificant increase of 39.2% was also observed for CSF samples treated with Triton X-100. There was not a clear effect of detergent treatment on total α-syn concentration due to a high level of variability.

   While oligomeric α-syn concentrations remained stable after up to six tube transfers, total α-syn concentration declined by an average of 9% per tube transfer, with a significant reduction in concentration observed after six transfers relative to CSF samples that were transferred once (36% reduction after 6 transfers; p=0.018).

   When freeze-thaw cycles were investigated, total α-syn concentration remained stable for up to 8 cycles relative to samples that underwent 1 cycle, while oligo α-syn concentrations decreased by 34% after four cycles, reaching significance after seven cycles (42% decline; p=0.021).

   Concentrations of total and oligomeric α-syn did not differ significantly among CSF samples that experienced a delay to frozen storage of 1-168 h at either room temperature or 4°C; concentrations also did not differ significantly when samples stored at room temperature and 4°C were compared.  The authors noted a gradual nonsignificant reduction (of 20%) in total α-syn concentration after 24 h of storage at room temperature.

   Biospecimens

   • Bodily Fluid - Cerebrospinal Fluid

   Preservative Types

   • Frozen

   Diagnoses:

   • Other diagnoses
   • Alzheimer's Disease

   Platform:

   Analyte	Technology Platform
   Protein	ELISA

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	Nunc: sterile, non-pyrogenic (1.8 ml)(Reference) Denville scientific: siliconized, enzyme free, DNA free (0.6 ml) Corning: sterile, RNase-free, DNase-free, non-pyrogenic (15 ml) Sarstedt: Sterile (0.5 ml) Eppendorf: Sterile, RNAse free, DNase free, pyrogen free, ATp free (1.5 ml) Extragene: non-sterile, DNase safe, RNase safe, pyrogen safe (1.5 ml) Costar: Non-sterile (2 ml)
   Biospecimen Acquisition	Pre-preservation condition	No detergent 0.5% Tween-20 0.5% Triton X-100 0.5% NP-40
   Storage	Freeze/thaw cycling	1 cycle 2 cycles 3 cycles 4 cycles 5 cycles 6 cycles 7 cycles 8 cycles
   Storage	Storage duration	1 h 2 h 4 h 24 h 72 h
   Storage	Storage temperature	Room temperature 4°C
   Storage	Specimen/sample transfer	0-6 tube transfers

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