Preanalytical Sample Handling and Sample Stability Testing for the Neurochemical Dementia Diagnostics

Abstract

Preanalytical sample handling and storage procedures play an extremely important role in reliably measuring neurochemical dementia diagnostics (NDD) biomarkers: Aβ(1-40), Aβ(1-42), Tau, and pTau181. To test different handling and storage conditions, the following protocols were applied: (a) storage at room temperature for one week, (b) deep-freezing and thawing up to three cycles, (c) deep-freezing, thawing and keeping under +4°C for two days before the analysis, and (d) long-term stability of a deeply frozen sample. Between the first and the seventh day of the storage at room temperature, the percentage of the concentrations (compared to the starting concentrations) fluctuated: 104.3-105.3, 97.6-93.2, 100.6-96.8, and 97.9-90.2 for Aβ(1-40), Aβ(1-42), Tau, and pTau181, respectively. Re-freezing cycles resulted in the percentage fluctuations of the concentrations: 101.1-105.5, 95.4-99.7, 98.3-100.0, and 100.5-101.4 for Aβ(1-40), Aβ(1-42), Tau, and pTau181, respectively. Keeping previously frozen/thawed samples under +4°C for two days resulted in the percentage differences of the concentrations: +15.9, +2.2, -1.1, and -0.1 for Aβ(1-40), Aβ(1-42), Tau, and pTau181, respectively. During long-term stability, the coefficients of linear correlation (R(2)) were: Aβ(1-40), 0.007; Aβ(1-42), 0.02; Tau, 0.011; and pTau181, 0.02, and the corresponding inter-assay coefficients of variation: 13.9%, 13.9%, 11.0%, and 10.7% for Aβ(1-40), Aβ(1-42), Tau, and pTau181, respectively. We conclude that the NDD biomarkers are relatively stable when the cerebrospinal fluid sample is kept at room temperature for about four days; one or two thawing/refreezing cycles do not profoundly affect the biomarkers concentrations, however three cycles result in increased unsystematic variation. The four biomarkers seem to be stable in a sample stored deeply frozen for more than two years.

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

“…Bjerke et al (6 ) stated that storage at room temperature for Ͼ24 h did not significantly affect A␤ concentrations in comparison to both fresh samples and storage at Ϫ80°C until analysis. Another study found relatively stable CSF biomarker concentrations even after 4 days, although unsystematic variation increased over time (18 ). In contrast to this, A␤ has been found to decrease during the first 48 h (11 ) or, more in line with the present study, even to increase within 24 h (14 ).…”

“…From long-term stability studies, it is already known that A␤ 1-42 concentrations in samples frozen at Ϫ80°C are stable for at least 2 years (6,18 ), as are concentrations of T-tau and P-tau 181P when stored at Ϫ20°C for at least for 2 and 4 years, respectively (12,18 ). A storage artifact is seen for cystatin C analysis after freezing at Ϫ20°C, but not at Ϫ80°C (24 ).…”

P1–178: CSF biomarker variability in the Alzheimer's Association quality control program

“…A small part of the overall variability was caused by within-run variability (CV, ,5%-10%, which is in agreement with previous reports and information from kit vendors regarding assay performance [21][22][23]). The within-laboratory longitudinal variability was larger (CV, 5%-19%), which is also in agreement with previous reports [8,24]. In analysis of variance, between-laboratory variability was a major contributor (19%-28%) to the overall variability.…”

Stability of amyloid‐β peptides in plasma and serum

“…Considerably, CSF Ab peptides have been reported to be more stable over a storage period of 2-5 days [15,17,18] as measured with distinct methods. These results are in line with our own observations measuring CSF Ab peptides under the conditions of the Ab-SDS-PAGE/immunoblot after samples were stored for up to 48 h. Thus, assay specifications of the Ab-SDS-PAGE/immunoblot are unlikely to account for the reported decrease in Ab peptides after sample storage in plasma and serum.…”