TY - JOUR
T1 - Pre-analytical factors influencing the stability of cerebrospinal fluid proteins
AU - Simonsen, Anja H
AU - Bahl, Justyna M C
AU - Danborg, Pia B
AU - Lindstrom, Veronica
AU - Larsen, Severin O
AU - Grubb, Anders
AU - Heegaard, Niels Henrik Helweg
AU - Waldemar, Gunhild
PY - 2013/5/5
Y1 - 2013/5/5
N2 - 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 4. h, 24. h 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.
AB - 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 4. h, 24. h 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.
U2 - 10.1016/j.jneumeth.2013.03.011
DO - 10.1016/j.jneumeth.2013.03.011
M3 - Journal article
C2 - 23537933
SN - 0165-0270
VL - 215
SP - 234
EP - 240
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
IS - 2
ER -