Impact of the transition from radioimmunoassay (RIA) to chemiluminescent enzyme immunoassay (CLEIA) for the measurement of plasma aldosterone concentration (PAC) on the diagnosis of primary aldosteronism (PA) via retrospective analyses in Okinawa, Japan.

In Japan, the traditional method for measuring plasma aldosterone concentration (PAC) was radioimmunoassay (RIA), which had several challenges, including poor traceability of certified reference materials and reduced detection sensitivity at low concentrations. To overcome these issues, a chemiluminescent enzyme immunoassay (CLEIA) for PAC measurement was introduced in April 2021 and the Japan Endocrine Society published new guidelines for primary aldosteronism (PA). This study aimed to evaluate the impact of the transition from RIA to CLEIA for PAC measurement on PA diagnosis. Data from 190 patients admitted to the Second Department of Internal Medicine, University of the Ryukyus Hospital, between April 2012 and March 2021 were analyzed. Patients who were diagnosed with PA underwent adrenal venous sampling. The PAC measured by RIA (PAC(RIA)) was converted to the estimated PAC measured by CLEIA (ePAC(CLEIA)) using a conversion formula. The present study evaluated the discordance rates in diagnoses based on screening (SC), captopril challenge test (CCT), saline infusion test (SIT), and diagnosis of PA between results judged by PAC(RIA) according to the previous guidelines and those judged by ePAC(CLEIA) according to the new guidelines. The results revealed discordant diagnosis rates of 6.4% for SC and 10.1% for CCT, with no discordance for SIT. The discordant diagnosis rate for PA was 3.7%. Our study reveals the challenges in establishing appropriate diagnostic criteria for PA using PAC(CLEIA) and highlights the demand for further research on provisionally positive categories.

A fit-for-purpose validation of a commercial radioimmunoassay for measurement of human peripheral oxytocin.

Oxytocin (OT) is a peptide hormone synthesized in the hypothalamus and released into systemic circulation or other areas of the brain. Its physiological roles include action as a hormone with stimulation of uterine contractions and that as a neuromodulator with involvement in social behaviors and regulation of mood. Its small size and low levels within biological matrices make it challenging to accurately measure. The goal of this study was to demonstrate the specificity of the antibody, sensitivity, and reproducibility of the Phoenix Pharmaceuticals (PP) OT radioimmunoassay (RIA) for use in human urine, serum, and saliva. Specificity of the antibody was assessed by high pressure liquid chromatography with ultraviolet (HPLC-UV) separation and assay of the fractions. Immunoreactivity was evaluated using the percent OT bound, and the fraction retention times were compared to the retention time of an intact OT standard to determine which fractions contained OT in the extracted samples. Reproducibility was assessed by running replicates of pools of each biomatrix over several assays. Sensitivity was assessed by repeated measurement of physiologically relevant low-concentration specimens. In all tested specimens the greatest reactivity in assay corresponded to the same fraction(s) as the OT standard. Only minimal reactivity was found in the other fractions, suggesting that in an unfractionated sample the antibody reacts mostly with intact OT. Reproducibility was acceptable for all specimens and the coefficient of variation (CV) ranged from 3.72 to 8.04% and 5.89-12.8%, for intra and inter-assay, respectively. The limits of quantitation (LOQ) were sufficient for measurement of normal values in urine (0.643 & 1.43 pg/mL), serum (1.90 pg/mL), and saliva pools (0.485 & 4.42 pg/mL). In conclusion, the PP OT RIA is specific and sensitive enough for reproducible measurement of intact OT in human peripheral biological matrices.

Comparison of three methods for the detection of antibodies against muscle-specific kinase.

OBJECTIVES: To compare 3 different methods for the detection of antibodies against muscle-specific kinase (MuSK). METHODS: MuSK antibody testing was performed in 237 serum samples by enzyme-linked immunosorbent assay (ELISA) and fixed cell-based assay (f-CBA-IFA). One hundred and forty-eight (148) of the sera had previously been tested by RIA during clinical testing: 47 MuSK antibody positive and 101 MuSK antibody negative. Of the MuSK RIA negative antibodies, 46 tested positive for other neural antibodies. Additionally, 89 sera were subsequently tested by all three methods: 70 healthy controls and 19 sera positive for other neural antibodies. RESULTS: Qualitative inter-assay agreement based on tiered RIA values was 100% for results of 1.00 nmol/L or greater by both methods; 81% and 94% for results between 0.21 and 0.99 nmol/L by ELISA and f-CBA-IFA, respectively; and 0% for results of 0.04-0.20 nmol/L by both methods. Negative results showed 100% agreement between RIA and both ELISA and f-CBA-IFA (n = 55). None of the controls positive for other neural autoantibodies or healthy controls were positive in any assay. CONCLUSION: Overall, excellent agreement was observed between the 3 methods used to detect antibodies against MuSK. Both the f-CBA-IFA and ELISA performed comparably to RIA and exhibited excellent overall accuracy for MuSK IgG detection, with the f-CBA-IFA demonstrating higher agreement between positive samples with the RIA than the ELISA without identifying false positives in the control samples. Advantages of non-radioactive methods for the detection of MuSK antibodies include reduced handling and disposal of hazardous materials, potential for automation and the reagents having a longer shelf-life, reducing costs associated with both workflow and lot validations. Thus, commercially available ELISA and transfected cell-based assays are viable alternatives to the traditional radioactive assay used for serologic determination of MuSK IgG.

Adrenal venous sampling criteria for chemiluminescent enzyme immunoassay as a preferable alternative to radioimmunoassay in primary aldosteronism.

Plasma aldosterone concentration (PAC) was routinely measured using radioimmunoassay (RIA); however, the RIA kit was discontinued in March 2021 in Japan. This study examined PAC conversion in adrenal venous sampling (AVS) and AVS criteria when measured using chemiluminescent enzyme immunoassay (CLEIA). PAC of 415 adrenal venous blood samples from AVS (including segmental AVS) of 63 patients with primary aldosteronism was measured using RIA (Spac-S aldosterone kit; Fujirebio Inc.) and CLEIA (Lumipulse Presto Aldosterone; Fujirebio Inc.). PAC of 70 AVS samples was also measured using liquid chromatography-mass spectrometry (LC-MS/MS, ASKA Pharma Medical Co., Ltd.). PAC conversion formulas were determined for each AVS sample assay. PAC measured using CLEIA was significantly correlated with that measured using RIA (correlation coefficient = 0.971). The PAC conversion formula was PAC (CLEIA) = PAC (RIA) × 0.772 - 1,199 pg/mL. The PAC of 14,000 pg/mL in RIA was equivalent to 9,613 pg/mL in CLEIA. PAC measured using CLEIA was also correlated with that measured using LC-MS/MS, and the PAC conversion formula was PAC (CLEIA, pg/mL) = 0.97 × PAC (LC-MS/MS, pg/mL) + 211. The inter-assay coefficient of variability (CV) was 1.1-1.3% and intra-assay CV was 1.0-1.7%, measured using CLEIA. The PAC conversion formula for AVS samples was obtained using CLEIA and RIA, and the conversion formula was different from that for peripheral blood. PAC values measured by CLEIA showed preferable accuracy and high concordance with those measured by LC-MS/MS, even in AVS samples. The study outcomes are useful for interpreting AVS results using non-RIA measurement methods.

Cancer and the Vasopressin Gene: Radioimmunoassay Values and Commentary on Copeptin as a Plasma Marker.

Background: Vasopressin gene expression has been demonstrated to be a common feature of all small-cell lung cancer (SCLC) and breast cancer. Provasopressin (ProVP) is a component of the cancer cell membrane and a likely target for treatment. However, a measurable fraction of this cancer provasopressin is also normally processed and products are released into the circulation. Vasopressin (VP) and vasopressin-associated human neurophysin (VP-HNP), two of three products of processing, were earlier shown to be reliable plasma markers for determining the presence of SCLC and monitoring response to treatment. Material and Methods: In this study, copeptin, the third product of provasopressin processing, was preliminarily evaluated as a plasma marker for SCLC or breast cancer using radioimmunoassay (RIA). Antibodies directed against the 18 residue C-terminal peptide fragment of copeptin were used to avoid interference from the large-carbohydrate component of this endogenous glycopeptide. Results: The levels of copeptin in 8 male and 6 female patients with SCLC before treatment ranged from 16 to 319 pmol/L, and these levels were elevated (>2.5 times) in 10 of 14 cases (70%) when compared with healthy volunteers (normal mean, 18 ± 6 pmol/L). Volunteer values for males were smaller than for females (15± 4 pmol/L and 20± 9 pmol/L), but numbers were small. Patients with breast cancer had plasma levels ranging from 12 to 68 pmol/L, with only three of the six elevated. Conclusion: While cancer patients displayed a wide range of plasma copeptin levels over 70% with SCLC and 50% with breast cancer had clearly elevated levels. This finding indicates that for such patients, plasma copeptin, like plasma VP and VP-HNP, could be used to detect disease. The control values found for healthy volunteers using our RIA were in a range predictable from established normal plasma levels of both VP and VP-HNP.

Experimental sickness reduces hypocretin receptor 1 expression in the lateral hypothalamus and ventral tegmental area of female mice.

Recent studies have focused on how sickness behaviours, including lethargy, are coordinated in the brain in response to peripheral infections. Decreased hypocretin (orexin) signalling is associated with lethargy and previous research suggests that hypocretin signalling is downregulated during sickness. However, there are studies that find increases or no change in hypocretin signalling during sickness. It is further unknown whether hypocretin receptor expression changes during sickness. Using lipopolysaccharide (LPS) to induce sickness in female mice, we investigated how LPS-injection affects gene expression of hypocretin receptors and prepro-hypocretin as well as hypocretin-1 peptide concentrations in brain tissue. We found that hypocretin receptor 1 gene expression was downregulated during sickness in the lateral hypothalamus and ventral tegmental area, but not in the dorsal raphe nucleus or locus coeruleus. We found no changes in hypocretin receptor 2 expression. Using a gene expression calculation that accounts for primer efficiencies and multiple endogenous controls, we were unable to detect changes in prepro-hypocretin expression. Using radioimmunoassay, we found no change in hypocretin-1 peptide in rostral brain tissue. Our results indicate that hypocretin receptor expression can fluctuate during sickness, adding an additional level of complexity to understanding hypocretin signalling during sickness.

An optimized radioimmunoassay for quantification of total serum thyroxine (T4) in fetal, neonatal, and pregnant rats.

Identifying xenobiotics that interrupt the thyroid axis has significant public health implications, given that thyroid hormones are required for brain development. As such, some developmental and reproductive toxicology (DART) studies now require or recommend serum total thyroxine (T4) measurements in pregnant, lactating, and developing rats. However, serum T4 concentrations are normally low in the fetus and pup which makes quantification difficult. These challenges can be circumvented by technologies like mass spectrometry, but these approaches are expensive and not always widely available. To demonstrate the feasibility of measuring T4 using a commercially available assay, we examine technical replicates of rat serum samples measured both by liquid chromatography mass spectrometry (LC/MS/MS) and radioimmunoassay (RIA). These samples were obtained from rats on gestational day 20 (dams and fetuses) or postnatal day 5 (pups), following maternal exposure to the goitrogen propylthiouracil (0-3 ppm) to incrementally decrease T4. We show that with assay modification, it is possible to measure serum T4 using low sample volumes (25-50 µL) by an RIA, including in the GD20 fetus exposed to propylthiouracil. This proof-of-concept study demonstrates the technical feasibility of measuring serum T4 in DART studies.

Method Validation and Establishment of Reference Intervals for an Insulin-like Growth Factor-1 Chemiluminescent Immunoassay in Cats.

Previously, radioimmunoassay (RIA) has been the only assay to measure insulin-like growth factor-1 (IGF-1) to diagnose hypersomatotropism (HS). Due to radiation concerns, availability, and the cost of IGF-1 RIA, validation of assays for automated analysers such as a chemiluminescent immunoassay (CLIA) is needed. The aim of this study was to validate a CLIA for measurement of feline IGF-1 (IMMULITE 2000® XPi, Siemens Medical Solutions Diagnostics, Malvern, PA, USA) compared to IGF1 RIA, establish reference interval (RI), and determine a cut-off value for diagnosis of HS in diabetic cats. Validation of assay performance included precision, linearity, and recovery studies. Right-sided RI was determined using surplus serum of 50 healthy adult cats. Surplus serum samples of diabetic cats with known IGF-1 concentration with (n = 32/68) and without HS (n = 36/68) were used for method comparison with RIA. The cut-off for diagnosis of HS was established using receiver operating characteristic (ROC) analysis. The intra-assay coefficient of variation (CV) was ≤4.7%, and the inter-assay CV was ≤5.6% for samples with low, medium, and high IGF-1 concentration. Linearity was excellent (R2 > 0.99). The correlation between CLIA and RIA was very high (rs = 0.97), with a mean negative bias for CLIA of 24.5%. The upper limit of RI was 670 ng/mL. ROC analysis showed an area under the curve of 0.94, with best cut-off for diagnosis of HS at 746 ng/mL (sensitivity, 84.4%; specificity, 97.2%). The performance of CLIA was good, and the RI and cut-off for HS diagnosis established in this study allow for CLIA to be used in routine work-up of diabetic cats.

Plasma Somatostatin Levels Increase during Scoliosis Surgery, but Not Herniated Disc Operations: Results of a Pilot Study.

Somatostatin (SST) released from capsaicin-sensitive sensory nerves in response to stimulation exerts systemic anti-inflammatory, analgesic actions. Its elevation correlates with the extent of tissue injury. We measured plasma SST alterations during spine operations (scoliosis and herniated disc) to determine whether its release might be a general protective mechanism during painful conditions. Sampling timepoints were baseline (1), after: soft tissue retraction (2), osteotomy (3), skin closure (4), the following morning (5). Plasma SST-like immunoreactivity (SST-LI) determined by radioimmunoassay was correlated with pain intensity and the correction angle (Cobb angle). In scoliosis surgery, postoperative pain intensity (VAS 2.) 1 day after surgery significantly increased (from 1.44 SEM ± 0.68 to 6.77 SEM ± 0.82, p = 0.0028) and positively correlated with the Cobb angle (p = 0.0235). The baseline Cobb degree negatively correlated (p = 0.0459) with the preoperative SST-LI. The plasma SST-LI significantly increased in fraction 3 compared to the baseline (p < 0.05), and significantly decreased thereafter (p < 0.001). In contrast, in herniated disc operations no SST-LI changes were observed in either group. The VAS decreased after surgery both in the traditional (mean 6.83 to 2.29, p = 0.0005) and microdiscectomy groups (mean 7.22 to 2.11, p = 0.0009). More extensive and destructive scoliosis surgery might cause greater tissue damage with greater pain (inflammation), which results in a significant SST release into the plasma from the sensory nerves. SST is suggested to be involved in an endogenous postoperative analgesic (anti-inflammatory) mechanism.

Establishment of a reference interval for 12-hour fasted serum gastrin concentration in adult dogs.

BACKGROUND: Adherence to traditional 24-h fasting periods for serum gastrin concentration in dogs can be challenging and may delay the institution of therapies for suspected hypergastrinemia. Peer-reviewed publications regarding serum gastrin reference intervals (RI) are lacking. Hypercalcemia is associated with hypergastrinemia in people; limited data exist in dogs. OBJECTIVE: The objective of the study was to generate a RI for a 12-h fasted serum gastrin concentration in dogs and to investigate whether correlations exist with age, weight, sex, and total calcium concentration. METHODS: Fifty-five healthy adult dogs (>1 year of age). The screening included: medical history, physical examination, CBC (15 dogs), and serum chemistry (55 dogs). Gastrin was measured via a commercial radioimmunoassay. The RI for 12-h fasted serum gastrin concentration was calculated according to the recommendations of the American Society for Veterinary Clinical Pathology. Additionally, data were evaluated for correlation with selected variables. RESULTS: The RI for serum gastrin following a 12-h fasting period was 15.1-78.9 ng/L with 90% confidence intervals for the lower and upper limits of 14.0-22.9 and 68.3-83.0 ng/L, respectively. A generalized linear model did not detect significant relationships between gastrin and age (P = 0.48), sex (P = 0.30), weight (P = 0.93), or total calcium concentration (P = 0.84). CONCLUSIONS: A 12-h fasted serum gastrin concentration RI has been established. Given the limited range of serum calcium concentrations in our healthy study population, additional investigations are needed to determine the effects of hypercalcemia on serum gastrin concentrations in dogs and for any potential clinical consequences thereof.

The mammalian gastro-intestinal tract is a NOT a major extra-pineal source of melatonin.

In 1992, a paper reported that the melatonin content of the rat duodenum was 24 000 ± 2000 pg/g tissue (range: 4000-100 000 pg/g) while the pineal melatonin content was 580 000 ± 36 000 pg/g. The data has been used for the last 30 years to infer that the gut produces 400 hundred times more melatonin than the pineal gland and that it is the source of plasma melatonin during the daytime. No-one has ever challenged the statement. In this review, evidence is summarised from the literature that pinealectomy eliminates melatonin from the circulation and that studies to the contrary have relied upon poorly validated immunoassays that overstate the levels. Similarly studies that have reported increases in plasma melatonin following tryptophan administration failed to account for cross reactivity of tryptophan and its metabolites in immunoassays. The most extraordinary observation from the literature is that in those studies that have measured melatonin in the gut since 1992, the tissue content is vastly lower than the original report, even when the methodology used could be overestimating the melatonin content due to cross reactivity. Using the more contemporary results we can calculate that rather than a 400:1 ratio of duodenum: pineal melatonin, a ratio of 0.05-0.19: 1 is likely. The gut is not a major extra-pineal source of melatonin; indeed it may well not produce any.

NANOCRINOLOGY.

We conceptualize and define nanocrinology as the science that studies the nanometric and subnanometric precision that operates in diagnostic and therapeutic endocrinology. It includes advanced generation assays, which can detect low concentrations of hormones, and modern drug delivery systems that allow more efficient delivery of endocrinotropic agents. Nanocrinology is a rapidly growing field of endocrinology, and we call for greater research and adoption of this science.

Advances and challenges in the measurement of 1,25-dihydroxyvitamin D: a comprehensive review.

Vitamin D has received significant attention from clinical societies, researchers, and the general population in recent years. While 25-hydroxyvitamin D (25(OH)D) is the most commonly-used biomarker of vitamin D status, 1α,25-dihydroxyvitamin D (1,25(OH)2D), its bioactive form, plays a critical role in regulating calcium and phosphorus homeostasis and is also involved in the immune system and cellular differentiation. Consequently, accurate measurements of 1,25(OH)2D can aid in the differential diagnosis of calcium-related disorders such as hypocalcemia in vitamin D-dependent rickets and hypercalcemia due to inappropriate increase of serum 1,25(OH)2D in granulomatous diseases. However, due to its lipophilicity and very low circulating concentration, the measurement of 1,25(OH)2D is particularly challenging. Over the past several decades, numerous efforts have been made to develop sensitive, specific, and practical laboratory methods for measuring 1,25(OH)2D. Methods using radioreceptor assay, radioimmunoassay, enzyme immunoassay, enzyme-linked immunosorbent assay, automated chemiluminescent immunoassay, and liquid chromatography-tandem mass spectrometry have been described. Each of these methods has unique advantages and limitations, and some are no longer used. Despite the sophisticated methods in use today, substantial variations between methods still exist. A concerted effort toward standardization of 1,25(OH)2D measurement is needed to ensure accurate and reliable results across laboratories and methods.

Optimization of a Quantitative Anti-Drug Antibodies against Infliximab Assay with the Liquid Chromatography-Tandem Mass Spectrometry: A Method Validation Study and Future Perspectives.

Monoclonal antibodies (mAbs), such as infliximab, are important treatment options for different diseases. Immunogenicity is a major risk, resulting in anti-drug antibodies (ADAs), being associated with adverse events and loss of response, influencing long-term outcomes. The development of ADAs against infliximab is primarily measured by immunoassays like radioimmunoassay (RIA). Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly utilized across different fields, this technique is currently not used for ADAs against infliximab measurements. Therefore, we developed the first LC-MS/MS method. Stable isotopically labeled infliximab antigen-binding fragments (SIL IFX F(ab')2) were used to bind and measure ADAs indirectly. Protein A magnetic beads were used to capture IgG, including ADAs, whereafter SIL IFX F(ab')2 was added for labeling. After washing, internal standard addition, elution, denaturation and digestion samples were measured by LC-MS/MS. Internal validation showed good linearity between 0.1 and 16 mg/L (R2 > 0.998). Sixty samples were used for cross-validation with RIA, and no significant difference between ADA concentrations was found. The methods had high correlation (R = 0.94, p < 0.001) and excellent agreement, intraclass correlation coefficient = 0.912 (95% confidence interval 0.858-0.947, p < 0.001). We present the first ADA against the infliximab LC-MS/MS method. The method is amendable for quantifying other ADAs, making it applicable as a template for future ADA methods.

Increased meal-induced neurotensin response predicts successful maintenance of weight loss - Data from a randomized controlled trial.

BACKGROUND: The gut derived anorexigenic hormone neurotensin (NT) is upregulated after bariatric surgery which may contribute to the sustained weight loss. In contrast, diet-induced weight loss is most often followed by weight regain. We therefore investigated whether diet-induced weight loss impacts levels of circulating NT in mice and humans and whether NT levels predicts body weight change after weight loss in humans. METHODS: In vivo mice study: Obese mice were fed ad-libitum or a restricted diet (40-60 % of average food intake) for 9 days to obtain similar weight loss as observed in the human study. At termination, intestinal segments, the hypothalamus and plasma were collected for histological, real time PCR, and radioimmunoassay (RIA) analysis. CLINICAL TRIAL: Plasma samples from 42 participants with obesity, completing an 8-week low-calorie diet in a randomized controlled trial, were analyzed. Plasma NT was measured by RIA at fasting and during a meal test before and after diet-induced weight loss and after one year of intended weight maintenance. RESULTS: In obese mice, food restriction-induced body weight loss of 14 % was associated with a 64 % reduction in fasting plasma NT (p < 0.0001). In the mouse duodenum (p = 0.07) and jejunum (p < 0.05), NT tissue concentration was decreased without tissue atrophy indicative of a physiological downregulation. In the mouse hypothalamus a downregulation of Pomc (p < 0.01) along with upregulation of Npy (p < 0.001) and Agrp (p < 0.0001) expression was found after restricted feeding in support of increased hunger after diet-induced weight loss. Therefore, we investigated the NT response in humans undergoing weight loss maintenance. In humans, similar to the mice, the low-calorie diet induced weight loss of 13 % body weight was associated with 40 % reduction in fasting plasma NT levels (p < 0.001). Meal-induced NT peak responses were greater in humans who lost additional weight during the 1 year maintenance phase compared to participants who regained weight (p < 0.05). CONCLUSION: Diet-induced weight loss decreased fasting plasma NT levels in both humans and mice with obesity, and regulated hunger-associated hypothalamic gene expression in mice. Meal-induced NT responses were greater in humans who lost additional weight during the 1 year maintenance phase compared to participants who regained weight. This indicates that increased peak secretion of NT after weight loss may contribute to successful maintenance of weight loss. CLINICAL TRIAL REGISTRATION NUMBER: NCT02094183.

Drug-tolerant detection of anti-drug antibodies in an antigen-binding assay using europium chelate fluorescence.

Accurate anti-drug antibody (ADA) measurements in patient sera requires dissociation of ADA-drug complexes combined with sensitive and specific ADA detection. Bridging type immunoassays are often used despite several disadvantages associated with this approach. A good drug-tolerant alternative is the acid-dissociation radioimmunoassay (ARIA), but this method is not easily implemented in most labs as specialized facilities are required for working with radioactive materials. We describe an innovative method for ADA detection that combines the advantages of antigen binding tests like the ARIA with the convenience of regular immunoassays. This acid-dissociation lanthanide-fluorescence immunoassay (ALFIA) involves dissociation of ADA-drug complexes, followed by binding to an europium-labeled drug derivative and subsequently an IgG pulldown on Sepharose beads. After europium elution, detection is achieved by measuring time-resolved fluorescence originating from europium chelate complexes. We measured anti-adalimumab ADA levels in sera of 94 rheumatoid arthritis patients using the ALFIA and showed this method to be highly drug tolerant, sensitive and specific for anti-adalimumab ADAs.

A novel fully-automated method to measure steroids in serum by liquid chromatography-tandem mass spectrometry.

Background: Steroids play a key role in numerous physiological processes. Steroid determination is a useful tool to explore various endocrine diseases. Because of its specificity, mass spectrometry is considered to be a reference method for the determination of steroids in serum compared to radioimmunoassay. This technology could progress towards more automation for the optimal organization of clinical laboratories and ultimately for the benefit of patients. Methods: A fully automated ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed and fully validated to determine five steroids in serum. Sample preparation was based on protein precipitation with filtration followed by online solid phase extraction. Chromatographic separation was performed using a biphenyl stationary phase. Results: The method was successfully validated according to European Medicine Agency guidelines. Coefficients of variation did not exceed, respectively, 8.4% and 8.1% for intra- and inter-assay precision. Method comparison with radioimmunoassay showed a proportional bias for all compounds, except for testosterone in men. Comparison with another LC-MS/MS method demonstrated acceptable concordance for all steroids, although a small bias was observed for androstenedione. Conclusion: The novelty of this method is that it has been fully automated. Automation provides benefits in traceability and allows significant savings in cost and time.

Repeated measures of hypocretin-1 in Danish and Italian patients with narcolepsy and in controls.

STUDY OBJECTIVES: The assay currently used worldwide to measure cerebrospinal fluid hypocretin-1 (CSF-hcrt-1) for diagnosing narcolepsy uses a competitive radioimmunoassay with polyclonal anti-hcrt-1 antibodies. This assay detects multiple hypocretin-1 immunoreactive species in the CSF that are all derived from full-length hcrt-1. We aimed to revalidate CSF-hcrt-1 cut-offs for narcolepsy type 1 (NT1) diagnosis and to evaluate temporal changes in CSF-hcrt-1 levels in patients suspected of having central hypersomnia. METHOD: We carried out a repeat lumbar puncture with a mean follow-up of 4.0 years, to measure CSF-hcrt-1 in patients suspected of having central hypersomnia in a follow-up study. Data from CSF samples of patients with NT1 and of controls without known hypersomnia, from the Italian-Stanford and Danish populations, were examined using a receiver-operating characteristic analysis. RESULTS: The optimal CSF-hcrt-1 cut-offs for identifying NT1 were 129 pg/ml and 179 pg/ml for the Italian-Stanford and Danish populations, respectively. The sensitivity was 0.93-0.99 and the specificity was 1. Follow-up lumbar puncture measurements of CSF-hcrt-1 were obtained from 73 patients. 30 of 32 patients with low CSF-hcrt-1 levels continued to be categorized as low, with an unaltered diagnosis; two patients showed a marked increase in CSF-hcrt-1, attaining normal values at follow-up. One of these patients relapsed to low CSF-hcrt-1 after follow-up. All 41 patients with normal CSF-hcrt-1 at baseline had normal CSF-hcrt-1 at follow-up. CONCLUSION: CSF-hcrt-1 measurement can provide an accurate test for diagnosing NT1, although it is important to validate the CSF-hcrt-1 cut-off for specific testing locations. Stable CSF-hcrt-1 levels support the already established prognosis of narcolepsy as permanent once the disorder has fully developed.