title: Chasing the Effects of Pre-Analytical Confounders - A Multicenter Study on CSF-AD Biomarkers.
abstract: BACKGROUND
  Core cerebrospinal fluid (CSF) biomarkers - Aβ42, Tau, and phosphorylated Tau (pTau) - have been recently incorporated in the revised criteria for Alzheimer's disease (AD). However, their widespread clinical application lacks standardization. Pre-analytical sample handling and storage play an important role in the reliable measurement of these biomarkers across laboratories.
  
  
  OBJECTIVE
  In this study, we aim to surpass the efforts from previous studies, by employing a multicenter approach to assess the impact of less studied CSF pre-analytical confounders in AD-biomarkers quantification.
  
  
  METHODS
  Four different centers participated in this study and followed the same established protocol. CSF samples were analyzed for three biomarkers (Aβ42, Tau, and pTau) and tested for different spinning conditions [temperature: room temperature (RT) vs. 4°C; speed: 500 vs. 2000 vs. 3000 g], storage volume variations (25, 50, and 75% of tube total volume), as well as freezing-thaw cycles (up to five cycles). The influence of sample routine parameters, inter-center variability, and relative value of each biomarker (reported as normal/abnormal) was analyzed.
  
  
  RESULTS
  Centrifugation conditions did not influence biomarkers levels, except for samples with a high CSF total protein content, where either non-centrifugation or centrifugation at RT, compared to 4°C, led to higher Aβ42 levels. Reducing CSF storage volume from 75 to 50% of total tube capacity decreased Aβ42 concentration (within analytical CV of the assay), whereas no change in Tau or pTau was observed. Moreover, the concentration of Tau and pTau appears to be stable up to five freeze-thaw cycles, whereas Aβ42 levels decrease if CSF is freeze-thawed more than three times.
  
  
  CONCLUSIONS
  This systematic study reinforces the need for CSF centrifugation at 4°C prior to storage and highlights the influence of storage conditions in Aβ42 levels. This study contributes to the establishment of harmonized standard operating procedures that will help reducing inter-lab variability of CSF-AD biomarkers evaluation.
authors.affiliation: Center for Neuroscience and Cell Biology (CNC), University of Coimbra , Coimbra , Portugal.
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Leitão
authors.affiliation: Center for Neuroscience and Cell Biology (CNC), University of Coimbra , Coimbra , Portugal ; Neurochemistry Laboratory, Neurology Department, Coimbra University Hospital , Coimbra , Portugal ; Faculty of Medicine, University of Coimbra , Coimbra , Portugal.
authors.forename: Inês
authors.initials: I
authors.lastname: Baldeiras
authors.affiliation: Neurology Department, Institute of Clinical Medicine, Kuopio University Hospital, University of Eastern Finland , Kuopio , Finland.
authors.forename: Sanna-Kaisa
authors.initials: SK
authors.lastname: Herukka
authors.affiliation: Neurology Department, Institute of Clinical Medicine, Kuopio University Hospital, University of Eastern Finland , Kuopio , Finland.
authors.forename: Maria
authors.initials: M
authors.lastname: Pikkarainen
authors.affiliation: Neurosurgery of NeuroCenter, Kuopio University Hospital , Kuopio , Finland.
authors.forename: Ville
authors.initials: V
authors.lastname: Leinonen
authors.affiliation: Danish Dementia Research Centre, Copenhagen University Hospital Rigshospitalet , Copenhagen , Denmark.
authors.forename: Anja Hviid
authors.initials: AH
authors.lastname: Simonsen
authors.affiliation: Neurobiology, Biochemistry and Molecular Biology Department, University Hospital of Lyon , Lyon , France ; UMR5292, BioRan, CNRS, INSERM U1028, University of Lyon 1 , Lyon , France ; Société Française de Biologie Clinique (SFBC), Alzheimer Biomarkers Group Co-Coordination , Lyon , France.
authors.forename: Armand
authors.initials: A
authors.lastname: Perret-Liaudet
authors.affiliation: Neurobiology, Biochemistry and Molecular Biology Department, University Hospital of Lyon , Lyon , France ; UMR5292, BioRan, CNRS, INSERM U1028, University of Lyon 1 , Lyon , France.
authors.forename: Anthony
authors.initials: A
authors.lastname: Fourier
authors.affiliation: Neurobiology, Biochemistry and Molecular Biology Department, University Hospital of Lyon , Lyon , France ; UMR5292, BioRan, CNRS, INSERM U1028, University of Lyon 1 , Lyon , France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Quadrio
authors.affiliation: Statistics and Research - Curva de Gauss, Training and Consulting , Canas de Senhorim , Portugal.
authors.forename: Pedro Mota
authors.initials: PM
authors.lastname: Veiga
authors.affiliation: Center for Neuroscience and Cell Biology (CNC), University of Coimbra , Coimbra , Portugal ; Neurochemistry Laboratory, Neurology Department, Coimbra University Hospital , Coimbra , Portugal ; Faculty of Medicine, University of Coimbra , Coimbra , Portugal.
authors.forename: Catarina Resende
authors.initials: CR
authors.lastname: de Oliveira
keywords: Alzheimer’s disease; BIOMARKAPD; biomarkers; cerebrospinal fluid; phosphorylated tau protein; standardized operating procedures; tau protein; β-amyloid