Importance and Impact of Preanalytical Variables on Alzheimer Disease Biomarker Concentrations in Cerebrospinal Fluid

Abstract

BACKGROUND Analyses of cerebrospinal fluid (CSF) biomarkers (β-amyloid protein, total tau protein, and hyperphosphorylated tau protein) are part of the diagnostic criteria of Alzheimer disease. Different preanalytical sample procedures contribute to variability of CSF biomarker concentrations, hampering between-laboratory comparisons. The aim of this study was to explore the influence of fractionated sampling, centrifugation, freezing temperature, freezing delay, and freeze–thaw cycles on CSF biomarker analyses. METHODS We studied fractionated sampling in sequential aliquots of lumbar CSF. Centrifuged and noncentrifuged samples from the same fraction were compared. CSF samples were subjected to different protocols (liquid nitrogen, −80 °C, and −20 °C; 24 h at 2–8 °C; and 24 and 48 h at room temperature). To study the influence of freeze–thaw cycles, samples were thawed up to 4 times and refrozen at −80 °C. CSF was collected in polypropylene tubes. We measured CSF biomarker concentrations with commercially available single-analyte Innotest assays. RESULTS CSF biomarker concentrations from non–blood-contaminated samples are not influenced by centrifugation or fractionated sampling. Freezing temperature and delayed storage can affect biomarker concentrations; freezing of CSF samples at −80 °C as soon as possible after collection is recommended. Consecutive freezing and thawing of CSF samples up to 3 times had little effect. CONCLUSIONS Temperature of freezing, delay until freezing, and freeze–thaw cycles significantly influence CSF biomarker concentrations, stressing the need for standard operating procedures for preanalytical sample handling. The differences observed in this study are, however, relatively small, and the impact on the clinical value of these CSF biomarkers needs to be determined.

Pre-analytical and analytical factors influencing Alzheimer's disease cerebrospinal fluid biomarker variability

“…In line with this, CSF t-Tau and p-Tau levels were reported to be significantly lower when CSF samples were frozen at −20°C instead of −80°C [16,34].…”

Preanalytical Variables and Alzheimer Disease Biomarker Concentrations in Cerebrospinal Fluid

“…However, no differences in A␤1-42 and T-tau concentrations between lumbar CSF fractions were found, consistent with previous studies (9 ) and also true for P-tau181P. Comparison of centrifuged and noncentrifuged samples from the same fractions revealed that centrifugation had no effect on CSF biomarker concentrations in macroscopically non-blood-contaminated samples (8 ). The practical implication is that centrifugation is not required for CSF biomarker analyses, and this likely is true, as the authors discuss, even when CSF is contaminated with blood (8 ).…”

“…Le Bastard et al concluded that it is possible to collect CSF in a single large volume or several smaller volumes from their observation that the total volume of CSF collected does not affect the concentrations of tau and amyloid proteins (8 ). Fractionated sampling of lumbar CSF has been postulated to be important, given that brain-derived proteins usually show a rostrocaudal concentration gradient, with higher concentrations in ventricular CSF compared to lumbar CSF, thereby implying that different volumes or fractionated sampling could generate differences in CSF biomarker concentrations.…”

“…The authors point out as a limitation of their study that physiological variability was limited through fractionated sampling in a relatively small number of patients that might not necessarily reflect daily clinical practice (8 ). The effect of the preanalytical variation might also depend on underlying brain pathology (AD vs non-AD dementias vs controls); thus replication in a larger and more heterogeneous population including a control group would be desirable (8 ).…”

“…The effect of the preanalytical variation might also depend on underlying brain pathology (AD vs non-AD dementias vs controls); thus replication in a larger and more heterogeneous population including a control group would be desirable (8 ).…”