Iron Horse has evaluated CSF samples from 106 subjects (45 ALS patients, 25 disease controls with a range of neurodegenerative conditions, and 36 healthy controls.CSF levels of pNfH, tau, C3, and C reactive protein (CRP) were measured. pNfH was significantly elevated in CSF from ALS subjects as compared with healthy and disease controls; there were no significant differences for CRP or tau. To further distinguish between ALS subjects and disease controls, data from cytoskeletal and inflammatory pathways were combined. A ratio of CSF levels of pNfH and C3 showed significant differences between ALS and both the disease and healthy control groups. Results were verified on a separate test set of CSF samples. Overall, the predictive pNfH/C3 ratio identified ALS with 87.3 % sensitivity and 94.6% specificity in a total of 71 ALS subjects, 52 disease control subjects, and 40 healthy subjects.