Pre-analytical stability of serum biomarkers for neurological disease: neurofilament-light, glial fibrillary acidic protein and contactin-1.

OBJECTIVES: Neurofilament-light (NfL), glial fibrillary acidic protein (GFAP) and contactin-1 (CNTN1) are blood-based biomarkers that could contribute to monitoring and prediction of disease and treatment outcomes in neurological diseases. Pre-analytical sample handling might affect results, which could be disease-dependent. We tested common handling variations in serum of volunteers as well as in a defined group of patients with multiple sclerosis (pwMS). METHODS: Sample sets from 5 pwMS and 5 volunteers at the outpatient clinic were collected per experiment. We investigated the effect of the following variables: collection tube type, delayed centrifugation, centrifugation temperature, delayed storage after centrifugation and freeze-thawing. NfL and GFAP were measured by Simoa and CNTN1 by Luminex. A median recovery of 90-110% was considered stable. RESULTS: For most pre-analytical variables, serum NfL and CNTN1 levels remained unaffected. In the total group, NfL levels increased (121%) after 6 h of delay at 2-8 °C until centrifugation, while no significant changes were observed after 24 h delay at room temperature (RT). In pwMS specifically, CNTN1 levels increased from additional freeze-thaw cycles number 2 to 4 (111%-141%), whereas volunteer levels remained stable. GFAP showed good stability for all pre-analytical variables. CONCLUSIONS: Overall, the serum biomarkers tested were relatively unaffected by variations in sample handling. For serum NfL, we recommend storage at RT before centrifugation at 2-8 °C up to 6 h or at RT up to 24 h. For serum CNTN1, we advise a maximum of two freeze-thaw cycles. Our results confirm and expand on recently launched consensus standardized operating procedures.

How to handle adsorption of cerebrospinal fluid amyloid ß (1-42) in laboratory practice? Identifying problematic handlings and resolving the issue by use of the Aß42/Aß40 ratio.

INTRODUCTION: We aimed to investigate factors defining amyloid ß (1-42) (Aß1-42) adsorption during preanalytical workup of cerebrospinal fluid (CSF). METHODS: CSF was transferred to new tubes ≤4 times. Variables tested were different polypropylene tube brands, volumes, CSF Aß1-42 concentrations, incubation times, pipettes, vortex intensities, and other CSF proteins, including hyperphosphorylated tau and Interleukin 1 Receptor Accessory Protein (IL-1RAcP). An enquiry assessed the number of transfers in current practice. RESULTS: In diagnostic practice, the number of transfers varied between 1 and 3. Every tube transfer resulted in 5% loss of Aß1-42 concentration, even 10% in small volumes. Adsorption was observed after 30 seconds and after contact with the pipette tip. Tube brand, vortexing, or continuous tube movement did not influence adsorption. Adsorption for Aß1-40 was similar, resulting in stable Aß1-42/Aß1-40 ratios over multiple tube transfers. DISCUSSION: We confirmed that adsorption of CSF Aß1-42 during preanalytical processing is an important confounder. However, use of the Aß1-42/Aß1-40 ratio overcomes this effect and can therefore contribute to increased diagnostic accuracy.

Effect of long-term storage in biobanks on cerebrospinal fluid biomarker Aß1-42, T-tau, and P-tau values.

INTRODUCTION: We studied the effect of long-term storage at -80°C on cerebrospinal fluid (CSF) biomarker levels. Our approach assumed consistency of mean biomarker levels in a homogenous Alzheimer's disease patient cohort over time. METHODS: We selected 148 Alzheimer's disease samples that had inclusion dates equally distributed over the years 2001 to 2013 from our biobank. The concentrations of CSF biomarkers, amyloid ß1-42 (Aß1-42), total tau (T-tau), and phosphorylated tau181 (P-tau), were measured with one enzyme-linked immunosorbent assay lot. Results were compared with historical results obtained at biobank inclusion. RESULTS: Linear regression analyses showed that the levels of CSF biomarkers, Aß1-42, T-tau, and P-tau, were not related to storage time at -80°C (ß = 0.015, 0.048, and 0.0016 pg/mL per day, not significant). However, the differences between remeasured concentrations of Aß1-42 and concentrations at biobank inclusion measured for more than 30 assay batches increased with increasing time difference. DISCUSSION: The levels of CSF biomarkers, Aß1-42, T-tau, and P-tau, did not significantly change during the maximum period of 12 years of storage at -80°C. Batch variation for Aß1-42 is a factor that should be controlled for when using historical cohorts.

Standardization of Assay Procedures for Analysis of the CSF Biomarkers Amyloid ß((1-42)), Tau, and Phosphorylated Tau in Alzheimer's Disease: Report of an International Workshop.

Large variation in assay performance and outcomes of CSF Aß1-42, total Tau (Tau), and phosphorylated Tau (pTau) (at amino acid 181) levels is observed between laboratories. The aim of this study was to assess the differences in assay procedures between several experienced international laboratories, as potential sources of error. 14 groups performed the Aß42, Tau, and pTau assays according to the guidelines of the manufacturer. Differences in analytical procedures between the laboratories were monitored. At least 23 items in assay procedures were identified that varied between the laboratories, including procedures for washing, pipetting, incubation, finishing, and sample handing. In general, the inter- and intra-assay variation between the groups was generally below 10% for all three assays. We concluded that 17 international centers that use the same assays for Aß42, Tau and pTau on a regular basis do not uniformly adhere to the procedures recommended by the manufacturer. For harmonization of intercenter results of these biomarkers standardization of protocols is highly needed.