The concentration of tau proteins in CSF increases as AD progresses. Total tau is a general marker of neuronal injury and is also elevated in other conditions such as traumatic brain injury, stroke and Creutzfeldt-Jakob disease. Phosphorylated tau (P-tau) is a specific indicator of neurofibrillary tangles. Tau phosphorylated at the position threonine 181 is the most studied form and serves as a marker for AD.
Determination of the triad of Aβ1-42/Aβ1-40 ratio, total tau and P-tau in CSF provides vital support in the diagnosis of AD and helps predict progression to dementia in patients with mild cognitive impairment.
Standardization and preanalytics
In recent years, there has been a concerted effort to standardize measurement of Alzheimer’s biomarkers in CSF. Aspects such as preanalytical sample handling, detection technology and reference materials can have a significant impact on the analytical results . These issues have been addressed in collaborative work between scientists, clinicians and industry partners such as EUROIMMUN, in particular as part of the Alzheimer’s Association Global Biomarker Standardization Consortium (GBSC). The GBSC recently published the first official guideline for preanalytical sample handling, which gives recommendations for collection, transport, handling and storage of CSF samples .
As Aβ peptides bind irreversibly to plastic and glass surfaces, contact of patient samples with consumables such as syringes, tubes and pipette tips must be minimized throughout the entire analysis. This limits adsorption and thus loss of the peptides and increases result reliability.
The guideline recommends collecting CSF by gravity drip directly into sample tubes with low-binding capacity. The sample should fill at least 50% of the tube volume to ensure a low ratio of contact surface to sample volume. Freezing of samples prior to transport is advisable to avoid unnecessary movement of liquid in the tubes. In the laboratory processing, transfers of samples into fresh tubes should be minimized to prevent further analyte loss. Moreover, repeated freeze-thaw cycles should be avoided as they can result in degradation of the analytes.
Notably, the effects of preanalytical factors are less severe when the recommended Aβ1-42/Aβ1-40 ratio is measured rather than Aβ1-42 alone . Since the isoforms Aβ1-42 and Aβ1-40 are subject to the impact factors to the same extent, measurement of the ratio helps to mitigate the preanalytical effects. This can be observed, for example, for sample vessel, storage, freeze-thaw cycles and sample volume (Fig. 2).
Aβ1-42 reference material
A lack of reference material previously hampered efforts to standardize CSF analyses in Alzheimer’s diagnostics. Certified reference material (CRM) for Aβ1-42 has recently been developed to enable more meaningful comparison of measurements between different laboratories and detection platforms. The CRM was developed by a working group comprising clinical laboratories in collaboration with the GBSC. The value was standardized by chromatography mass spectrometry. As a member of the working group, EUROIMMUN has ensured that its Aβ1-42 assays are aligned to the reference material.
Aβ peptides and tau proteins can be reliably measured in CSF samples using methods such as enzyme-linked immunosorbent assay (ELISA) or chemiluminescence immunoassay (ChLIA). Key requirements for the assays are high specificity and accuracy, as well as standardized protocols for simple processing and automatability.
In collaboration with ADx Neurosciences, a leader in Alzheimer’s diagnostics, EUROIMMUN has developed a range of quantitative ELISAs and ChLIAs that provide robust and highly reproducible measurement of Aβ1-42, Aβ1-40, total tau or P-tau. The assays are based on highly specific monoclonal antibodies, which are coated onto microplates (ELISA) or magnetic particles (ChLIA). The incubation procedures for each method are identical, so that the different parameters can easily be processed in parallel. The ELISA procedure takes approximately 5 hours, while the ChLIA analysis takes just 25 minutes until the first result. Lyophilized calibrators and controls supplied in the kits enhance convenience and ensure high precision.
Fully automated processing of the tests increases the efficiency and standardization of the analyses. The EUROIMMUN ELISAs can be processed on devices such as the EUROLabWorkstation ELISA or the EUROIMMUN Analyzer I or I-2P. The CHLIAs can be processed on the benchtop random access instruments IDS-i10 or IDS-iSYS Multi-Discipline Automated System.