Hypocretin measurement: shelf age of radioimmunoassay kit, but not freezer time, influences assay variability

• Authors: Glenda Keating, Emory University; Donald Bliwise, Emory University; Prabhjyot Saini, Emory University; David Rye, Emory University; Lynn Marie Trotti, Emory University
• Journal Title: Scandinavian Journal of Clinical and Laboratory Investigation
• Volume: Volume 77, Number 5
• Publisher: Taylor & Francis: STM, Behavioural Science and Public Health Titles | 2017-01-01, Pages 390-393
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.1080/00365513.2017.1325928
• Subjects: Health Sciences, Medicine and Surgery
• Keywords: Science & Technology; Life Sciences & Biomedicine; Medicine, Research & Experimental; Research & Experimental Medicine; Assay; cerebrospinal fluid; immunoassay; narcolepsy; sleep wake disorders; CEREBROSPINAL-FLUID; NARCOLEPSY

Abstract

The hypothalamic peptide hypocretin 1 (orexin A) may be assayed in cerebrospinal fluid to diagnose narcolepsy type 1. This testing is not commercially available, and factors contributing to assay variability have not previously been comprehensively explored. In the present study, cerebrospinal fluid hypocretin concentrations were determined in duplicate in 155 patient samples, across a range of sleep disorders. Intra-assay variability of these measures was analyzed. Inter-assay correlation between samples tested at Emory and at Stanford was high (r=0.79, p<0.0001). Intra-assay correlation between samples tested in duplicate in our center was also high (r=0.88, p<0.0001); intra-assay variability, expressed as the difference between values as a percentage of the higher value, was low at 9.4% (SD=7.9%). Although both time the sample spent in the freezer (r=0.16, p=0.04) and age of the kit used for assay (t=3.64, p=0.0004) were significant predictors of intra-kit variability in univariate analyses, only age of kit was significant in multivariate linear regression (F=4.93, p=0.03). Age of radioimmunoassay kit affects intra-kit variability of measured hypocretin values, such that kits closer to expiration exhibit significantly more variability.