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Measurement of estradiol, estrone, and testosterone in postmenopausal human serum by isotope dilution liquid chromatography tandem mass spectrometry without derivatization.
Steroids. 2015 Jan 21;
Authors: Wooding KM, Hankin JA, Johnson CA, Chosich JD, Baek SW, Bradford AP, Murphy RC, Santoro N
BACKGROUND: A high-throughput, sensitive, specific, mass spectrometry-based method for quantitating estrone (E1), estradiol (E2), and testosterone (T) in postmenopausal human serum has been developed for clinical research. The method consumes 100 ul human serum for each measurement (triplicates consume 300 ul) and does not require derivatization. We adapted a commercially available 96-well plate for sample preparation, extraction, and introduction into the mass spectrometer on a single platform.
METHODS: Steroid extraction from serum samples and mass spectrometer operational parameters were optimized for analysis of estradiol and subsequently applied to other analytes. In addition to determining the limit of detection (LOD) and limit of quantitation (LOQ) from standard curves, a serum LOQ (sLOQ) was determined by addition of known steroid quantities to serum samples. Mass spectrometric method quantitative data were compared to results using a state-of-the-art ELISA (enzyme-linked immunosorbent assay) using stored serum samples from menopausal women.
RESULTS: The LOD, LOQ, sLOQ was (0.1 pg, 0.3 pg, 1 pg/ml) for estrone, (0.3 pg, 1 pg, 3 pg/ml) for estradiol, and (0.3 pg, 1 pg, 30pg/ml) for testosterone, respectively. Mass spectrometry accurately determined concentrations of E2 that could not be quantified by immunochemical methods. E1 concentrations measured by mass spectrometry were in all cases significantly lower than the ELISA measurements, suggesting immunoreactive contaminants in serum may interfere with ELISA. The testosterone measurements broadly agreed with each other in that both techniques could differentiate between low, medium and high serum levels.
CONCLUSIONS: We have developed and validated a scalable, sensitive assay for trace quantitation of E1, E2 and T in human serum samples in a single assay using sample preparation method and stable isotope dilution mass spectrometry.
PMID: 25617740 [PubMed - as supplied by publisher]
Population-based evaluation of retinal nerve fiber layer, retinal ganglion cell layer, and inner plexiform layer as a diagnostic tool for glaucoma.
Invest Ophthalmol Vis Sci. 2014 Dec;55(12):8428-38
Authors: Springelkamp H, Lee K, Wolfs RC, Buitendijk GH, Ramdas WD, Hofman A, Vingerling JR, Klaver CC, Abràmoff MD, Jansonius NM
PURPOSE: We determined the glaucoma screening performance of regional optical coherence tomography (OCT) layer thickness measurements in the peripapillary and macular region, in a population-based setting.
METHODS: Subjects (n = 1224) in the Rotterdam Study underwent visual field testing (Humphrey Field Analyzer) and OCT of the macula and optic nerve head (Topcon 3-D OCT-1000). We determined the mean thicknesses of the retinal nerve fiber layer (RNFL), retinal ganglion cell layer (RGCL), and inner plexiform layer for regions-of-interest; thus, defining a series of OCT parameters, using the Iowa Reference Algorithms. Reference standard was the presence of glaucomatous visual field loss (GVFL); controls were subjects without GVFL, an intraocular pressure (IOP) of 21 mm Hg or less, and no positive family history for glaucoma. We calculated the area under the receiver operating characteristics curve (AUCs) and the sensitivity at 97.5% specificity for each parameter.
RESULTS: After excluding 23 subjects with an IOP > 21 mm Hg and 73 subjects with a positive family history for glaucoma, there were 1087 controls and 41 glaucoma cases. Mean RGCL thickness in the inferior half of the macular region showed the highest AUC (0.85; 95% confidence interval [CI] 0.77-0.92) and sensitivity (53.7%; 95% CI, 38.7-68.0%). The mean thickness of the peripapillary RNFL had an AUC of 0.77 (95% CI, 0.69-0.85) and a sensitivity of 24.4% (95% CI, 13.7-39.5%).
CONCLUSIONS: Macular RGCL loss is at least as common as peripapillary RNFL abnormalities in population-based glaucoma cases. Screening for glaucoma using OCT-derived regional thickness identifies approximately half of those cases of glaucoma as diagnosed by perimetry.
PMID: 25414193 [PubMed - indexed for MEDLINE]