- Email: [email protected]
QUANTIFICATION OF HEPCIDIN-25 IN HUMAN CEREBROSPINAL FLUID: TECHNICAL FEASIBILITY AND BIOLOGICAL RELEVANCE IN NEURODEGENERATIVE DISEASES
P354
Poster Presentations: Sunday, July 16, 2017
College London, London, United Kingdom; 4Institute of Child Health, University College London, London, United Kingdom. Contact e-mail: [email protected] Background: Frontotemporal dementia (FTD) is a progressive,
neurodegenerative disorder with clinical and pathological heterogeneity. The main clinical FTD phenotypes are behavioural variant FTD (bvFTD), semantic dementia (SD) and progressive nonfluent aphasia (PNFA). One of the key clinical characteristics of bvFTD is disturbance in eating behaviour, which can be helpful in diagnosing bvFTD and differentiating it from Alzheimer’s disease (AD). The aim of this study was to develop a hypothalamic peptide panel, focusing on measures known to be involved in appetite regulation, to gain a better understanding of the pathophysiology of FTD, and potentially leading to better fluid biomarkers. Methods: A peptide multiplex panel of 13 hypothalamic and 9 peripheral appetite regulating peptides was developed on a liquid chromatography coupled tandem mass spectrometry platform. Concentrations were measured in the CSF of the three clinical FTD phenotypes (bvFTD n¼9, SD n¼9, PNFA n¼4) as well as AD (n¼4) and healthy controls (n¼6) and compared using non-parametric statistical tests. Results: In five of the hypothalamic peptides there was a significant difference (expressed as pmol/300 ml) between controls and at least one of the FTD groups (p<0.05): neuropeptide W levels were significantly higher in all three groups: bvFTD (0.029), SD (0.034) and PNFA (0.039), controls (0.012); cerebellin was decreased in bvFTD (0.586) and SD (0.591) [controls 0.945], and cocaine-amphetamine regulated transcript was decreased in PNFA (0.359) and SD (0.359) [controls 0.575]; corticotropinreleasing hormone was decreased in bvFTD (0.007) [controls 0.011] and galanin was increased in PNFA (0.107) [controls 0.059]. There was also a trend of decreased levels of pro-orexin in bvFTD. There were also significant differences in two of the peripheral peptides in PNFA (insulin-like growth factor 1 and pancreatic polypeptide). Conclusions: This pilot study shows changes in concentration of a substantial proportion of the hypothalamic peptides within the CSF in the FTD groups compared to controls. Further exploration on a larger clinically defined cohort will enable understanding of the differences in hypothalamic peptides in FTD and investigate whether such a panel could be used as a biomarker in FTD disease diagnosis, prognosis or stratification.
Montpellier, Montpellier, France. Contact e-mail: constance.delaby@ inserm.fr Background: Hepcidin, the main iron metabolism regulator is classically measured in patients’ serum for diagnosis and follow-up of numerous disorders (anemia, hemochromatosis, chronic inflammation, cancer.). However, assessing hepcidin-25 levels in other fluids such as the cerebrospinal fluid (CSF) may present biological relevance. In particular, this might be of high interest for the diagnosis, follow-up or treatment of neuronal disorders linked to iron metabolism or toxicity, including neurodegenerative diseases such as Parkinson or Alzheimer’s disease. We describe in the present work an LC–MS/ MS assay for the quantification of hepcidin-25 in the CSF. Our results confirm the validity of our method as a means of measuring this hormone in human CSF samples. Moreover, our preliminary results show potential clinical relevance of CSF hepcidin-25 determination for the diagnosis and follow-up of patients with iron-related brain disorders. Methods: Hepcidin-25 was quantified through LC-MS/MS method. Analytical validation included determination of LOQ (0.1 ng/ml), repeatability, intermediate precision, recovery and linearity (up to 25 ng/ml). Hepcidin-25 was subsequently quantified in human CSF samples and its concentration ranged from 0.21 to 3.54 ng/ml. Samples originated from patients suffering various neurological disorders who signed informed consent for research. In addition, we assessed in these patients CSF levels of the following biomarkers: Ab 1-42 and 1-40 (Fujirebio and Euroimmun kits), Tau and p-Tau (181) (Fujirebio kits). Finally, we compared our MS method to the high sensitive ELISA kit (DRG-diagnostics) for CSF hepcidin-25 quantification. Results: Using our LC-MS/MS method, human CSF hepcidin-25 concentration ranged from 0.21 to 3.54 ng/ml and was thus detectable in all samples. It is noteworthy that CSF hepcidin could also be detected using the commercial high sensitive Hepcidin ELISA assay and that significant differences in CSF hepcidin were observed between different diagnoses. Conclusions: We developed a specific and technically feasible method able to detect and quantify hepcidin-25 in human CSF. This method, based on LC–MS/MS approaches, has been analytically validated and appears suitable to quantify hepcidin-25 in various pathological situations, in a range of 0.21–3.54 ng/ml. Preliminary results illustrate potential relevance of CSF hepcidin-25 quantification for clinical follow-up of iron-related neuronal disorders.
P1-274
P1-273
QUANTIFICATION OF HEPCIDIN-25 IN HUMAN CEREBROSPINAL FLUID: TECHNICAL FEASIBILITY AND BIOLOGICAL RELEVANCE IN NEURODEGENERATIVE DISEASES
Constance Delaby1,2, Pauline Bros1,3, Jer^ome Vialaret4, Cyndi Catteau1, Amandine Moulinier1, Alexia Picas1, Vincent Delatour3, Audrey Gabelle1,2,5, Yves Dauvilliers2,6, Christian Geny2,7, Christophe Hirtz1,2, Sylvain Lehmann1,2, 1Laboratoire de Biochimie Proteomique Clinique - CHU Montpellier, Montpellier, France; 2Universite de Montpellier, Montpellier, France; 3Laboratoire National de Metrologie et d’Essais, Paris, France; 4Laboratoire de Biochimie Proteomique Clinique - CHU Montpellier, Montpellier, France; 5Centre Memoire Ressources Recherche, Departement de Neurologie - CHU Gui de Chauliac, Montpellier, France; 6Centre de Reference Sommeil et Narcolepsie, CHU Gui de Chauliac, Montpellier, France; 7Departement de Geriatrie CHU
DEVELOPMENT OF A 3-PLEX DIGITAL IMMUNOASSAY FOR DETECTION OF AMYLOID BETA1-42, AMYLOID BETA1-40 AND TAU IN BLOOD AND CSF SAMPLES
Lei Chang, Dandan Shan, David Wilson, Quanterix Corporation, Lexington, MA, USA. Contact e-mail: [email protected] Background: Multiplexed measurement of Amyloid beta1-42
(Ab42), amyloid beta1-40 (Ab40) and total tau in serum, plasma and CSF continues to be challenging, especially for normal healthy individuals. Methods: A multiplex sandwich immunoassay was developed for Ab42, Ab40 and total tau for the Simoa HD-1 Analyzer. The Analyzer performs a 2-step sandwich immunoassay, transfers labeled capture beads to an array of microwells, and interrogates the wells for presence of enzyme label. A single labeled analyte molecule provides sufficient fluorescence in 30 seconds to be detected. The concentration of target is then interpolated from a calibration curve. The multiplex was evaluated for sensitivity,
Poster Presentations: Sunday, July 16, 2017
College London, London, United Kingdom; 4Institute of Child Health, University College London, London, United Kingdom. Contact e-mail: [email protected] Background: Frontotemporal dementia (FTD) is a progressive,
neurodegenerative disorder with clinical and pathological heterogeneity. The main clinical FTD phenotypes are behavioural variant FTD (bvFTD), semantic dementia (SD) and progressive nonfluent aphasia (PNFA). One of the key clinical characteristics of bvFTD is disturbance in eating behaviour, which can be helpful in diagnosing bvFTD and differentiating it from Alzheimer’s disease (AD). The aim of this study was to develop a hypothalamic peptide panel, focusing on measures known to be involved in appetite regulation, to gain a better understanding of the pathophysiology of FTD, and potentially leading to better fluid biomarkers. Methods: A peptide multiplex panel of 13 hypothalamic and 9 peripheral appetite regulating peptides was developed on a liquid chromatography coupled tandem mass spectrometry platform. Concentrations were measured in the CSF of the three clinical FTD phenotypes (bvFTD n¼9, SD n¼9, PNFA n¼4) as well as AD (n¼4) and healthy controls (n¼6) and compared using non-parametric statistical tests. Results: In five of the hypothalamic peptides there was a significant difference (expressed as pmol/300 ml) between controls and at least one of the FTD groups (p<0.05): neuropeptide W levels were significantly higher in all three groups: bvFTD (0.029), SD (0.034) and PNFA (0.039), controls (0.012); cerebellin was decreased in bvFTD (0.586) and SD (0.591) [controls 0.945], and cocaine-amphetamine regulated transcript was decreased in PNFA (0.359) and SD (0.359) [controls 0.575]; corticotropinreleasing hormone was decreased in bvFTD (0.007) [controls 0.011] and galanin was increased in PNFA (0.107) [controls 0.059]. There was also a trend of decreased levels of pro-orexin in bvFTD. There were also significant differences in two of the peripheral peptides in PNFA (insulin-like growth factor 1 and pancreatic polypeptide). Conclusions: This pilot study shows changes in concentration of a substantial proportion of the hypothalamic peptides within the CSF in the FTD groups compared to controls. Further exploration on a larger clinically defined cohort will enable understanding of the differences in hypothalamic peptides in FTD and investigate whether such a panel could be used as a biomarker in FTD disease diagnosis, prognosis or stratification.
Montpellier, Montpellier, France. Contact e-mail: constance.delaby@ inserm.fr Background: Hepcidin, the main iron metabolism regulator is classically measured in patients’ serum for diagnosis and follow-up of numerous disorders (anemia, hemochromatosis, chronic inflammation, cancer.). However, assessing hepcidin-25 levels in other fluids such as the cerebrospinal fluid (CSF) may present biological relevance. In particular, this might be of high interest for the diagnosis, follow-up or treatment of neuronal disorders linked to iron metabolism or toxicity, including neurodegenerative diseases such as Parkinson or Alzheimer’s disease. We describe in the present work an LC–MS/ MS assay for the quantification of hepcidin-25 in the CSF. Our results confirm the validity of our method as a means of measuring this hormone in human CSF samples. Moreover, our preliminary results show potential clinical relevance of CSF hepcidin-25 determination for the diagnosis and follow-up of patients with iron-related brain disorders. Methods: Hepcidin-25 was quantified through LC-MS/MS method. Analytical validation included determination of LOQ (0.1 ng/ml), repeatability, intermediate precision, recovery and linearity (up to 25 ng/ml). Hepcidin-25 was subsequently quantified in human CSF samples and its concentration ranged from 0.21 to 3.54 ng/ml. Samples originated from patients suffering various neurological disorders who signed informed consent for research. In addition, we assessed in these patients CSF levels of the following biomarkers: Ab 1-42 and 1-40 (Fujirebio and Euroimmun kits), Tau and p-Tau (181) (Fujirebio kits). Finally, we compared our MS method to the high sensitive ELISA kit (DRG-diagnostics) for CSF hepcidin-25 quantification. Results: Using our LC-MS/MS method, human CSF hepcidin-25 concentration ranged from 0.21 to 3.54 ng/ml and was thus detectable in all samples. It is noteworthy that CSF hepcidin could also be detected using the commercial high sensitive Hepcidin ELISA assay and that significant differences in CSF hepcidin were observed between different diagnoses. Conclusions: We developed a specific and technically feasible method able to detect and quantify hepcidin-25 in human CSF. This method, based on LC–MS/MS approaches, has been analytically validated and appears suitable to quantify hepcidin-25 in various pathological situations, in a range of 0.21–3.54 ng/ml. Preliminary results illustrate potential relevance of CSF hepcidin-25 quantification for clinical follow-up of iron-related neuronal disorders.
P1-274
P1-273
QUANTIFICATION OF HEPCIDIN-25 IN HUMAN CEREBROSPINAL FLUID: TECHNICAL FEASIBILITY AND BIOLOGICAL RELEVANCE IN NEURODEGENERATIVE DISEASES
Constance Delaby1,2, Pauline Bros1,3, Jer^ome Vialaret4, Cyndi Catteau1, Amandine Moulinier1, Alexia Picas1, Vincent Delatour3, Audrey Gabelle1,2,5, Yves Dauvilliers2,6, Christian Geny2,7, Christophe Hirtz1,2, Sylvain Lehmann1,2, 1Laboratoire de Biochimie Proteomique Clinique - CHU Montpellier, Montpellier, France; 2Universite de Montpellier, Montpellier, France; 3Laboratoire National de Metrologie et d’Essais, Paris, France; 4Laboratoire de Biochimie Proteomique Clinique - CHU Montpellier, Montpellier, France; 5Centre Memoire Ressources Recherche, Departement de Neurologie - CHU Gui de Chauliac, Montpellier, France; 6Centre de Reference Sommeil et Narcolepsie, CHU Gui de Chauliac, Montpellier, France; 7Departement de Geriatrie CHU
DEVELOPMENT OF A 3-PLEX DIGITAL IMMUNOASSAY FOR DETECTION OF AMYLOID BETA1-42, AMYLOID BETA1-40 AND TAU IN BLOOD AND CSF SAMPLES
Lei Chang, Dandan Shan, David Wilson, Quanterix Corporation, Lexington, MA, USA. Contact e-mail: [email protected] Background: Multiplexed measurement of Amyloid beta1-42
(Ab42), amyloid beta1-40 (Ab40) and total tau in serum, plasma and CSF continues to be challenging, especially for normal healthy individuals. Methods: A multiplex sandwich immunoassay was developed for Ab42, Ab40 and total tau for the Simoa HD-1 Analyzer. The Analyzer performs a 2-step sandwich immunoassay, transfers labeled capture beads to an array of microwells, and interrogates the wells for presence of enzyme label. A single labeled analyte molecule provides sufficient fluorescence in 30 seconds to be detected. The concentration of target is then interpolated from a calibration curve. The multiplex was evaluated for sensitivity,