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 degrees 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 degrees 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.

Automated summary

The study found that most pre-analytical handling variations had little effect on NfL and CNTN1 levels, while GFAP showed good stability under all pre-analytical variables. For sample handling, it is recommended to store serum NfL at room temperature for up to 6 hours or at room temperature for up to 24 hours, and to perform a maximum of two freeze-thaw cycles for serum CNTN1.