Hypocretin-1 measurements in cerebrospinal fluid using radioimmunoassay: within and between assay reliability and limit of quantification

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van der Hoeven, Adrienne Elisabeth¹  van Waaij, Kevin¹  Bijlenga, Denise¹  Roelandse, Frederik Willem Cornelis³  Overeem, Sebastiaan⁴  Bakker, Jaap Adriaan³  Fronczek, Rolf¹  Lammers, Gert Jan¹ 

[1] Department of Neurology, Leiden University Medical Center;Sleep-Wake Center;Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center;Sleep Medicine Center

关键词: orexin-A;    narcolepsy;    intra-assay coefficient of variation;    inter-assay coefficient of variation;   

DOI  :  10.1093/sleep/zsac103

学科分类：生理学

来源: American Academy of Sleep Medicine

【 摘 要 】

Study Objectives The most sensitive and specific investigative method for the diagnosis of narcolepsy type 1 (NT1) is the determination of hypocretin-1 (orexin-A) deficiency (≤110 pg/mL) in cerebrospinal fluid using a radioimmunoassay (RIA). We aimed to assess the reliability of the Phoenix Pharmaceuticals hypocretin-1 RIA, by determining the lower limit of quantification (LLOQ), the variability around the cutoff of 110 pg/mL, and the inter- and intra-assay variability.Methods Raw data of 80 consecutive hypocretin-1 RIAs were used to estimate the intra- and inter-assay coefficient of variation (CV). The LLOQ was established and defined as the lowest converted concentration with a CV <25%; the conversion is performed using a harmonization sample which is internationally used to minimize variation between RIAs.Results The mean intra-assay CV was 4.7%, while the unconverted inter-assay CV was 28.3% (18.5% excluding 2 outliers) and 7.5% when converted to international values. The LLOQ was determined as 27.9 pg/mL. The intra-assay CV of RIAs with lower specific radioactive activity showed a median of 5.6% (n = 41, range 1.6%–17.0%), which was significantly higher than in RIAs with higher specific activity (n = 36; median 3.2%, range 0.4%–11.6%, p = .013). The CV around the 110 pg/mL cutoff was <7%.Conclusions Hypocretin-1 RIAs should always be harmonized using standard reference material. The specific activity of an RIA has a significant impact on its reliability, because of the decay of 125I radioactivity. Values around the hypocretin-1 cut-off can reliably be measured. Hypocretin-1 concentrations below 28 pg/mL should be reported as “undetectable” when measured with the Phoenix Pharmaceuticals RIA.

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