Towards a unified protocol for handling of CSF before β-amyloid measurements

Background: Widespread implementation of Alzheimer's disease biomarkers in routine clinical practice requires the establishment of standard operating procedures for pre-analytical handling of cerebrospinal fluid (CSF). Methods: Here, CSF collection and storage protocols were optimized for measurements of beta-amyloid (A beta). We investigated the effects of (1) storage temperature, (2) storage time, (3) centrifugation, (4) sample mixing, (5) blood contamination, and (6) collection gradient on CSF levels of A beta. For each study participant, we used fresh CSF directly collected into a protein low binding (LoB) tube that was analyzed within hours after lumbar puncture (LP) as standard of truth. A beta 42 and A beta 40 were measured in de-identified CSF samples using EUROIMMUN and Mesoscale discovery assays. Results: CSF A beta 42 and A beta 40 were stable for at least 72 h at room temperature (RT), 1 week at 4 degrees C, and 2 weeks at -20 degrees C and - 80 degrees C. Centrifugation of non-blood-contaminated CSF or mixing of samples before the analysis did not affect A beta levels. Addition of 0.1-10% blood to CSF that was stored at RT without centrifugation led to a dose- and time-dependent decrease in A beta 42 and A beta 40, while A beta 42/A beta 40 did not change. The effects of blood contamination were mitigated by centrifugation and/or storage at 4 degrees C or - 20 degrees C. A beta levels did not differ between the first to fourth 5-ml portions of CSF. Conclusions: CSF can be stored for up to 72 h at RT, 1 week at 4 degrees C, or at least 2 weeks at either - 20 degrees C or - 80 degrees C before A beta measurements. Centrifugation of fresh non-blood-contaminated CSF after LP, or mixing before analysis, is not required. In case of visible blood contamination, centrifugation and storage at 4 degrees C or - 20 degrees C is recommended. After discarding the first 2 ml, any portion of up to 20 ml of CSF is suitable for A beta analysis. These findings will be important for the development of a clinical routine protocol for pre-analytical handling of CSF.