Cerebrospinal fluid (CSF) is normally a clear, colorless body fluid found throughout the central nervous system (CNS) including in the brain and spinal cord. Up to 500 mL of CSF is produced per day in humans by neuroepithelial multiciliate ependymal cells of the choroid plexuses located in the ventricles of the brain. Only about 125–150 mL is present at any one time due to continual re-absorption by the arachnoid granulations (Wright et al., 2012). CSF provides buoyancy for the brain providing basic mechanical and immunological protection, homeostasis, nutrient distribution and clearing of waste in the CNS.

Once believed to be immune privileged, the CNS is now known to show classic characteristics of inflammation when injured or infected. Cytokines produced in the brain by microglial and mast cells and dispersed through CSF, act in a similar fashion to those in peripheral blood by inducing chemokines and adhesion molecules and recruiting additional immune cells in a protective inflammatory response to external pathogens or cell damage (Rauf et al., 2022). Thus CSF is an ideal body fluid for the discovery of potential biomarkers for neuroinflammatory and other disorders of the CNS including multiple sclerosis (Schain and Kreisl, 2017; Khaibullin et al., 2017), Alzheimer's disease (Schain and Kreisl, 2017; Rani et al., 2022), Parkinson's disease, amyotrophic lateral sclerosis (Schain and Kreisl, 2017; Liu et al., 2020), Neurosarcoidosis (Lacomis, 2011; Kidd, 2020) and encephalitis (Ellul and Solomon, 2018; Kennedy, 2004). Peripheral blood cytokine concentrations have been associated with a variety of inflammatory and autoimmune conditions with a growing list of immunomodulating therapies including Interferon alpha and beta for hepatitis C and multiple sclerosis treatment (Jasyk and Siednienko, 2021), Interleukin 2 in cancer treatment and autoimmune diseases (Raeber et al., 2023), and Interferon gamma to treat chronic granulomatous disease (CGD) (Åhlin et al., 1999; Marciano et al., 2004) and osteopetrosis (Key et al., 1992; Tang et al., 2018). Specific cytokine blockers, including the anti-TNF therapeutics infliximab, adalimumab, etanercept, certolizumab pegol and golimumab have been breakthrough treatments for Crohn's disease (Adegbola et al., 2018; Levin et al., 2016), ulcerative colitis (Park and Jeen, 2015; Pugliese et al., 2017), rheumatoid arthritis (Ma and Xu, 2013; Chen et al., 2006) and ankylosing spondylitis (Coates et al., 2010; McLeod et al., 2007). Less is known, however, about cytokine levels in CSF for these diseases and treatment, and while serum and plasma cytokine testing is widely available in clinical laboratories, with established normal reference ranges, cytokine testing in CSF is more limited and if performed, usually reported as an unvalidated specimen type with no accompanying normal reference range. In this study, we validated CSF as a specimen type and established normal reference intervals for multiple cytokines and markers.

We employed a multiplexed bead-based assay previously validated to measure 13 cytokines/markers in serum and plasma for the validation of CSF as a specimen type and for establishing reference intervals in normal CSF specimens. The assay can quantitate concentrations for TH1 type cytokines: IFNγ, IL12, IL2; TH2 type: IL4, IL5, IL10, IL13; Monokines: IL1β, IL6, IL8, TNFα; as well as IL17 and IL2 receptor using only 25 μL of specimen.