Fluid-based biomarkers of amyotrophic lateral sclerosis: recent advances and future prospects

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder with no definitive cure. The absence of specific diagnostic biomarkers leads to diagnostic delays, hindering early intervention and management. This review provides a critical appraisal of fluid-based biomarkers for ALS across multiple sources-cerebrospinal fluid (CSF), blood, urine, saliva, and tears-with emphasis on their diagnostic and prognostic potential, limitations, and readiness for clinical translation. While neurofilaments (NfL, pNfH) are well-established as sensitive indicators of neuroaxonal injury and are increasingly used as prognostic and pharmacodynamic markers in clinical trials, they lack disease specificity. Biomarkers reflecting ALS-specific pathology, such as TDP-43 species and C9orf72 dipeptide repeat proteins (DPRs), show promise but remain in early validation stages with limited multicenter data. Emerging markers from non-invasive sources (urine p75ECD, salivary chromogranin A, tear metabolomics) offer potential for repeated sampling but require rigorous external validation before clinical adoption. To address current gaps, we introduce a standardized evidence grading framework (Tier 1-3) and a comprehensive reporting template for biomarker studies, including explicit performance metrics (AUC, sensitivity, specificity, confidence intervals) and validation status. We also propose minimum reporting standards for study design, pre-analytical variables, and statistical rigor, modeled on REMARK guidelines. A roadmap for biomarker validation and a cross-fluid comparison matrix are provided to guide future research. Despite considerable progress, significant challenges remain, including biological heterogeneity, pre-analytical variability, and insufficient external validation. Future efforts should prioritize multicenter prospective studies, assay harmonization, ethical frameworks for early diagnosis, and integration of emerging technologies such as artificial intelligence and digital twins. Fluid-based biomarkers, while not yet replacing clinical evaluation, are essential tools for accelerating drug development, enabling patient stratification, and moving toward personalized medicine in ALS.