P 90 Clinical feasibility of novel ultra-sensitive multiplex NfL, Tau, UCHL-1 and GFAP assay for evaluation of neuro-axonal and glial damage in serum samples of patients with multiple sclerosis using reduced cost protocol

Abstract

Introduction: Multiple sclerosis (MS) is an inflammatory disease of CNS with neuro-axonal injury as the key morphological correlate for long-term disability. Novel ultra-sensitive single molecule array (SiMoA) based detection of neurofilament light (NfL) and other neuroglial proteins is being considered as a promising tool for evaluation of disease activity 1 . However, clinical applicability is hampered by 1) considerable per-sample-costs, 2) scarcity of inter-laboratory comparisons and 3) absence of accepted cut-off values with good sensitivity and specificity. In this small single-center study, we aimed to a) show feasibility and clinical validity of an adapted reduced-cost protocol and to b) test sensitivity and specificity of an available 4-plex assay for NfL, Tau, UCHL-1 and GFAP in patients vs. controls and active vs. stable disease from a real-world data set of patients with MS and age matched healthy controls. Patients & Methods: We prospectively recruited serum samples from 114 adult patients with relapsing remitting MS (RRMS) including 42 with radiological/clinical active and 72 patients with stable disease as well as 70 healthy controls. Serum samples were measured with Neurology 4-Plex assay on fully automated HD-X-Analyzer (Quanterix) using standard and reduced cost protocols (measuring the serum samples in duplicates and singlicates). We determined precision, inter- and intra-assay variation, inter-laboratory comparisons, ROC-analysis, specificity and sensitivity using standard and modified protocols. Results: Using our reduced-cost approach, we confirmed inter- and intra-assay as well as inter-laboratory comparison coefficients of variations across relevant concentration levels of <10% for NfL. We reproduced higher mean serum NfL concentrations in patients with active RRMS (12.1pg/ml [95%CI 10.1-21.6]) compared to stable RRMS (7.1 pg/ml [95%CI 6.3-8.6], p<0,0001) and healthy controls (9.2 pg/ml [95%CI 8.2-10.3] p<0,0001) and significantly reduced per-sample-costs while applying our adapted approach. Serum tau (p<0.0001) but not GFAP and UCH-L1 concentrations were higher in RRMS versus controls. ROC-analysis (AUC=76.9±0,05) using a cut-off with maximal sensitivity showed a sensitivity of 70% and a specificity of 76% for active RRMS versus stable disease. The combinations of biomarkers showed no additional benefit over serum NfL concentration alone for prediction of disease activity. Conclusion: Using fully-automated analysis pipelines, reduced cost protocols are feasible and precise and could facilitate clinical application of serum neuronal biomarkers. While NfL discriminates fairly well between RRMS and healthy controls, sensitivity and specificity for active disease is lacking. Further prospective studies should evaluate the use of serial NfL measurements to determine clinical validity in detecting active RRMS.