Alzheimer’s disease and its biomarkers in CSF
Alzheimer’s disease is a chronic neurodegenerative disorder considered the most common cause of dementia in older people. Although clinical symptoms still form the core of diagnosis, imaging and cerebrospinal fluid biomarkers now play an important role in the diagnostic process. Two important neuropathological features of Alzheimer’s are the extracellular deposition of beta-amyloid (Aβ) plaques and hyperphosphorylated tau protein in neurofibrillary tangles, processes that can be detected through biomarkers in the cerebrospinal fluid (CSF): Aβ1-42 and Aβ1 -40, total Tau (t-Tau) and phospho Tau (p-Tau).
In this regard, people with Alzheimer’s have a characteristic CSF biochemical profile consisting of a decrease in the Aβ1-42/Aβ1-40 ratio together with an increase in the concentration of t-Tau and p-Tau. However, it is important to properly control the conditions under which CSF samples are collected, processed, and stored because these factors have been shown to be of great importance in analytical results. The most accepted collection/storage protocol requires that once the lumbar puncture is performed, the samples are quickly stored in ultra-low freezers at -80ºC until the moment of analysis. This makes it difficult for some centers with limited laboratory infrastructure to do so.
The objective of our study, recently published in the journal Clinical Chemistry and Laboratory Medicine and entitled “Importance of cerebrospinal fluid store conditions for the Alzheimer’s disease diagnostics on an automated platform” was to determine if storage conditions influence the quantifications of biomarkers of Alzheimer’s in CSF. This study has been done in collaboration between the Memory Unit (Neurology) and the Biochemistry service of the Hospital de Sant Pau.
What have we done in this study?
In this study, we obtained CSF samples from eleven patients who underwent lumbar puncture at the Sant Pau Memory Unit in order to confirm or rule out the diagnosis of Alzheimer’s. The samples were collected and processed following the established protocols. Regarding storage, part of the samples was divided and stored following different conditions of temperature (room temperature, 4ºC, -20ºC, -80ºC) and time (between 1-2 days and 15-16 days).
We measured and compared the values of the Alzheimer’s markers (Aβ1-42, Aβ1-40, T-Tau and P-Tau) in each sample under the different conditions using the automated analysis technique implemented at the Hospital de Sant Pau.
Main results
Our study showed that conditions involving storage at 4ºC for 1-2 days, 8-9 days, or 15-16 days were associated with decreased Aβ fractions, especially for Aβ1-42 and to a lesser extent for Aβ1- 40. Although the use of the Aβ1-42/Aβ1-40 ratio could partially compensate for this decrease, statistical differences could still be observed. Freezing conditions at -20°C or -80°C did not affect the Aβ fractions.
The results also showed that storing CSF samples up to 48 hours at room temperature and then freezing them at -80°C did not significantly affect the results of these markers, and that storing samples at -20°C (standard freezer) was equivalent to storing them at -80ºC (ultra-freezer), at least for 15-16 days.
Regarding the concentrations of p-Tau and t-Tau, they were not affected by any of the storage conditions evaluated, reinforcing the idea of their stability in different conditions and storage time.
Relevance of the study
This study confirms once again that amyloid-beta protein is very sensitive to preanalytical manipulation, but also that this is partially compensated using the Aβ1-42/Aβ1-40 ratio.
Our results allow us to conclude that centers with limited laboratory infrastructure can collect and store samples following simpler pre-analytical conditions and then send them to another reference center for analysis without impacting the results of the analyses.