Abstract
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. 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. 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. 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. 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.
Highlights
Core cerebrospinal fluid (CSF) biomarkers – Aβ42, Tau, and phosphorylated Tau – have been recently incorporated in the revised criteria for Alzheimer’s disease (AD)
Data were reanalyzed, testing the influence of the following co-variates: “Center,” “Clinical Group,” “CSF Total Protein,” and “RBC count”, “Biomarker Classification” in normal or abnormal for each protein according to each laboratory cut-offs
Samples with high Total protein (TP) (>44 mg/dL) had increased levels of Aβ42 if centrifuged at room temperature (RT) (571.5 ± 261.8) compared to 4°C (549.4 ± 238.0), whereas samples with normal TP had higher Aβ42 levels when centrifuged at 4°C (527.2 ± 226.2, 4°C vs. 498.4 ± 237.2, RT)
Summary
Core cerebrospinal fluid (CSF) biomarkers – Aβ42, Tau, and phosphorylated Tau (pTau) – have been recently incorporated in the revised criteria for Alzheimer’s disease (AD). Cerebrospinal fluid (CSF) biomarkers, Aβ42, Tau protein, and phosphorylated Tau (pTau), are frequently assessed for their proven value as hallmarks of initial and coursing neuropathological events in Alzheimer’s disease (AD) [1, 2]. Besides giving clues to pathogenic mechanisms of the disease, biomarkers can favor therapeutics development by signaling desired effects of drugs in phase I–II clinical trials, allowing inclusion of early cases to longitudinal studies and even identifying sub-groups of patients in order to tailor treatment [11, 12]
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