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

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.

Keywords:

• Assay
• cerebrospinal fluid
• immunoassay
• narcolepsy
• sleep wake disorders

Acknowledgements

We gratefully acknowledge Drs Emmanuel Mignot and Ling Lin (Stanford Center for Sleep Sciences and Medicine) for performing hypocretin measurements for the samples tested in duplicate at Stanford.

Disclosure statement

Dr Bliwise reports personal fees from Ferring Pharmaceuticals and Merck, outside the submitted work. Dr Rye reports personal fees from Jazz Pharmaceuticals, UCB Pharma, Flamel Technologies, and Xenoport Inc., outside the submitted work. Dr Rye has a US patent application (US 20110028418A1) pending. Dr Trotti reports fees to her institution from Jazz Pharmaceuticals and Balance Therapeutics, outside the submitted work. Dr Keating and Mr Saini declare no competing interests.

Additional information

Funding

This work was supported by the National Institutes of Health under grants K23 NS083748 (LMT) and R01 NS089719 (DBR); and the Mind Science Foundation (DBR).