Quantification of blood glial fibrillary acidic protein using a second-generation microfluidic assay. Validation and comparative analysis with two established assays

Abstract Background Increased levels of glial fibrillary acidic protein (GFAP) in blood have been identified as a valuable biomarker for some neurological disorders, such as Alzheimer’s disease and multiple sclerosis. However, most blood GFAP quantifications so far were performed using the same bead-based assay, and to date a routine clinical application is lacking. Methods In this study, we validated a novel second-generation (2 nd gen) Ella assay to quantify serum GFAP. Furthermore, we compared its performance with a bead-based single molecule array (Simoa) and a homemade blood GFAP assay in a clinical cohort of neurological diseases, including 210 patients. Results Validation experiments resulted in an intra-assay variation of 10%, an inter-assay of 12%, a limit of detection of 0.9 pg/mL, a lower limit of quantification of 2.8_pg/mL, and less than 20% variation in serum samples exposed to up to five freeze-thaw cycles, 120_hours at 4 °C and room temperature. Measurement of the clinical cohort using all assays revealed the same pattern of GFAP distribution in the different diagnostic groups. Moreover, we observed a strong correlation between the 2 nd gen Ella and Simoa (r=0.91 (95% CI: 0.88 - 0.93), p<0.0001) and the homemade immunoassay (r=0.77 (95% CI: 0.70 - 0.82), p<0.0001). Conclusions Our results demonstrate a high reliability, precision and reproducibility of the 2 nd gen Ella assay. Although a higher assay sensitivity for Simoa was observed, the new microfluidic assay might have the potential to be used for GFAP analysis in daily clinical workups due to its robustness and ease of use. What is already known on this topic Blood glial fibrillary acidic protein (GFAP) levels are an emerging biomarker for diagnosing, prognosis and treatment monitoring for AD, MS and other neurological disorders. However, so far, the application in clinical routine remains a challenge. What this study adds This study validated a novel, easy-to-use second-generation microfluidic assay for the quantitative measurement of blood GFAP. Moreover, its performance was compared to two other GFAP immunoassays, including single molecule array. How this study might affect research, practice or policy This study proved the reliability, precision and reproducibility of the novel second-generation microfluidic assay, which might be more easily implemented in daily clinical routine analyses and therefore facilitates the application of GFAP as a biomarker for neurological diseases.