title: Pre-analytical factors influencing the stability of cerebrospinal fluid proteins.
abstract: Cerebrospinal fluid (CSF) is a potential source for new biomarkers due to its proximity to the brain. This study aimed to clarify the stability of the CSF proteome when undergoing pre-analytical factors. We investigated the effects of repeated freeze/thaw cycles, protease inhibitors and delayed storage for 4h, 24h or 14 days at -20°C, 4°C and room temperature (RT) after centrifugation compared with our standard practice of two hours at RT before placing the samples in an -80°C environment. The results were obtained using immunoassays for amyloid-beta 1-42 (Aβ42), tau, phosphorylated tau (P-tau) and cystatin C and using surface-enhanced laser desorption/ionisation time-of-flight (SELDI-TOF) mass spectrometry for proteomic profiling. Tau and P-tau were susceptible to repeated freeze/thaw cycles while SELDI-TOF analysis produced eight significant peaks and additional artefact peaks from samples with added protease inhibitors. Delayed storage for different durations and in different temperatures produced six significant SELDI-TOF peaks. Aβ42 and tau were susceptible to increased temperatures and the duration before storage, whereas P-tau and cystatin C were not. Transthyretin and several of its isoforms were found using SELDI-TOF and were susceptible to freeze/thaw cycles and to increased temperature and length of time prior to storage. We recommend that CSF should be collected and centrifuged immediately after sampling and prior to storage at -80°C without the addition of protease inhibitors. Freeze/thawing should be avoided because of the instability of tau, P-tau and transthyretin. Standardised CSF sampling, handling and storage for biomarker research are essential for accurately comparing the results obtained by different studies and institutions.
authors.affiliation: Memory Disorders Research Group, Department of Neurology, Rigshospitalet, Copenhagen University Hospital, Denmark. anja.hviid.simonsen@rh.regionh.dk
authors.forename: Anja H
authors.initials: AH
authors.lastname: Simonsen
authors.forename: Justyna M C
authors.initials: JM
authors.lastname: Bahl
authors.forename: Pia B
authors.initials: PB
authors.lastname: Danborg
authors.forename: Veronica
authors.initials: V
authors.lastname: Lindstrom
authors.forename: Severin O
authors.initials: SO
authors.lastname: Larsen
authors.forename: Anders
authors.initials: A
authors.lastname: Grubb
authors.forename: Niels H H
authors.initials: NH
authors.lastname: Heegaard
authors.forename: Gunhild
authors.initials: G
authors.lastname: Waldemar
mesh_terms: D016229:Amyloid beta-Peptides / Q000134:cerebrospinal fluid; D002556:Cerebrospinal Fluid Proteins / Q000032:analysis* / Q000187:drug effects; D003072:Cognition Disorders / Q000134:cerebrospinal fluid*; D055316:Cystatin C / Q000134:cerebrospinal fluid; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005615:Freezing; D006801:Humans; D008297:Male; D010446:Peptide Fragments / Q000134:cerebrospinal fluid; D011480:Protease Inhibitors / Q000494:pharmacology; D013048:Specimen Handling; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013696:Temperature; D013997:Time Factors; D016875:tau Proteins / Q000134:cerebrospinal fluid
chemical_list: D016229:Amyloid beta-Peptides; D002556:Cerebrospinal Fluid Proteins; D055316:Cystatin C; D010446:Peptide Fragments; D011480:Protease Inhibitors; C075222:amyloid beta-protein (1-42); D016875:tau Proteins