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 cut-off 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, 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 cut-off 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 cutt-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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