Analyzing cerebrospinal fluid (CSF) biomarkers—including β-amyloid protein (Aβ1–42), total tau protein (T-tau), and hyperphosphorylated tau protein (P-tau181P)—is part of the process of diagnosing Alzheimer’s disease (AD). Preanalytical sample procedures can contribute to the variability of CSF biomarker concentrations, which can affect between-laboratory comparisons. A new study published in Clinical Chemistry and conducted by Nathalie Le Bastard, Peter Paul De Deyn, and Sebastiaan Engelborghs, sought to explore the influences of fractionated sampling, centrifugation, freezing temperature, freezing delay, and freeze-thaw cycles on biomarker analyses.
“The goals of using biomarkers for dementia are to enable presymptomatic diagnosis and to monitor disease progression and response to therapeutic interventions, often still in an investigative setting,” wrote Christopher M. Florkowski in an accompanying editorial. “As in other situations, interpretation should always be in full clinical context. Thus postanalytical as well as preanalytical and analytical factors need to be taken into consideration.”
The concentrations of AD CSF biomarkers can vary considerably, and the source of variation can often come from patient selection, as well as from preanalytical, analytical, or postanalytical interpretations of biomarker data. Confounding factors tied to the preanalytical process include lumbar puncture (LP) procedures, tubes used for sample collection and storage, and sample storage and handling.
The authors found that centrifugation and fractionated sampling did not have a great effect on AD biomarker concentrations, but the temperature of freezing, delayed storage, and freeze-thaw cycles did have an impact.
Based on the findings, the authors made the following recommendations:
- Fractionated sampling. Collect CSF in 1 large volume (for division later) or several smaller volumes.
- Centrifugation: If atraumatic samples are used, centrifugation is not needed.
- Freezing temperature: Freezing at -80 °C is a general recommendation for both long-term and short-term storage.
- Freezing delay: Minimize the delay in freezing.
- Freeze–thaw cycles: The standard procedure is one freeze–thaw cycle right before analysis; 1 or 2 additional freeze–thaw cycles are also allowed.
“Ultimately, the responsibility falls to authors of guidelines and individual laboratories to stipulate what level of preanalytical stringency is required, balancing clinical needs with practical expediency,” Florkowski wrote. “Improved comprehension of the role of preanalytical variables in AD biomarkers based on carefully conducted studies, like the study of Le Bastard et al., is a helpful step forward.”