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PubMed2001

Automated sample preparation and gas chromatographic-mass spectrometric analysis of urinary androgenic anabolic steroids.

This paper presents an automated method for extracting anabolic agents from urine samples for their GC-MS analysis by selected-ion monitoring. The sample preparation was carried out in a Hewlett-Packard 7686 SPE PrepStation system. Each 0.6-ml aliquot was hydrolyzed, extracted, dried and trimethylsilyl (TMS) derivatized in a 2-ml vial without any hands-on labor. When sample preparation was finished 2 microl of the extract was injected into the gas chromatograph by split (1:10) mode. Due to the small amount of free space in the 2-ml vials for handling the sample, parameters like time of hydrolysis, type of shaking, number of extractions and some TMS derivatization parameters had to be adjusted to achieve the best recovery for all of the compounds in the screening. Manual and automated sample preparation schemes were compared in terms of linearity, precision, accuracy, limit of detection and recovery data. When large concentrations were analyzed using the automated method no carry-over effect was observed.

Anabolic Agents↗

PubMed1999

Transfer from manual to automated sample preparation: a case study.

A manual sample preparation for a controlled release capsule formulation has been converted to an automated sample preparation. Each step of the manual sample preparation was evaluated as to its feasibility for automation in terms of precision, carryover, filter selection and other critical issues. Although most steps of the manual method were easily translated to the automated procedure, certain 'simple' details such as filter selection, sample storage, and the conversion from volumetric to gravimetric measurements needed closer investigation.

Automation↗

PubMed2001

Automation of the synthesis of highly concentrated 188Re-MAG3 for intracoronary radiation therapy.

We have developed an efficient method and an automated synthetic system for the preparation of highly concentrated 188Re-MAG3. Routine production of 188Re-MAG3 for use in intracoronary radiation therapy was performed by compressed air driven semi-automated shielded system. 188Re-MAG3 was prepared with a commercial kit and reducing agents, purified and concentrated by C18 Sep-Pak cartridges to desired radioactivity and volume. Using this automated system, reproducible radiolabeling yields of 80-85% were obtained.

Angioplasty, Balloon, Coronary↗

PubMed1997

[Comparative validation of manual and automated methods for mixing and volume control of total blood samples].

During blood collection, agitation and volume limitations are critical to ensure thorough mixing of the blood with the anticoagulant and obtention of the predetermined volume. These 2 factors are essential to prevent blood activation and to obtain well standardized blood products. The objective of this study was to compare the quality of the blood collected using 2 types of collection method: tripping of a scale at a predetermined volume limit of 450 mL in the presence of manual agitation, and the 3 blood collection monitors currently available in France. A minimum of 100 collection procedures was performed for each of the 4 methods tested. Results were found to be equivalent using either the manual or the automated procedures with regard to both the accuracy and reproducibility of the blood volumes obtained and the collection times and flow rates. The characteristics of the red blood cell concentrates, platelet concentrates and plasma units prepared from the first 30 collections of each group were assessed and compared to regulatory requirements. The quality of all these products was found to be comparable to that currently observed at quality control and no product was rejected at the release control for reasons of poor collection. An assessment of the practicability of the different methods showed that the automated devices are subject to practical difficulties involving transport and battery loading. In addition, the cost of this equipment is approximately 5 times higher than that of the scales. In conclusion, the results of this study show that in our hands, no significant advantage could be expected from the use of automated blood collection monitors as compared to simple scales with manual mixing. These results further raise the question of the applicability to labile blood products of the comparative validations currently accepted in the pharmaceutical industry, in order to allow the use of correctly validated alternative methods.

Automation↗

PubMed2005

Automation and validation of DNA-banking systems.

DNA banking is one of the central capabilities on which modern genetic research rests. The DNA-banking system plays an essential role in the flow of genetic data from patients and genetics researchers to the application of genetic research in the clinic. Until relatively recently, large collections of DNA samples were not common in human genetics. Now, collections of hundreds of thousands of samples are common in academic institutions and private companies. Automation of DNA banking can dramatically increase throughput, eliminate manual errors and improve the productivity of genetics research. An increased emphasis on pharmacogenetics and personalized medicine has highlighted the need for genetics laboratories to operate within the principles of a recognized quality system such as good laboratory practice (GLP). Automated systems are suitable for such laboratories but require a level of validation that might be unfamiliar to many genetics researchers. In this article, we use the AstraZeneca automated DNA archive and reformatting system (DART) as a case study of how such a system can be successfully developed and validated within the principles of GLP.

Automation↗

PubMed2001

Automated detection of tracé alternant during sleep in healthy full-term neonates using discrete wavelet transform.

OBJECTIVE: To develop an automated procedure for scoring neonatal sleep states using signal processing which are based on visual pattern recognition techniques. METHODS: We are developing an automated computer system to study relationships among multiple non-cerebral physiologic measures and brain activity in newborn infants, and are evaluating the usefulness of a number of different time-frequency domain transforms as potential diagnostic tools. RESULTS: Wavelet transforms yield excellent results in the detection of all twenty tracé alternant quiet sleep segments for 6 full-term healthy infants. CONCLUSIONS: We suggest that this method will be useful for the automated detection of neonatal sleep states, and may help delineate when sleep cycle disturbances occur on either an environmental or disease basis. More accurate physiologic descriptions of neonatal state may improve the clinician's ability to assess functional brain organization for a given post-conceptional age as well as document functional brain maturation at progressively older corrected ages.

Algorithms↗

PubMed2003

An automated method to determine the transcranial magnetic stimulation-induced contralateral silent period.

BACKGROUND: The transcranial magnetic stimulation (TMS)-induced contralateral silent period (CSP) refers to a period of interruption of voluntary muscle activity measured in tonically active muscles. The length of the CSP is generally interpreted to reflect cortical inhibition. The determination of the return of voluntary motor activity is typically accomplished via visual inspection of the electromyography (EMG) waveform and may be subject to inaccuracy on the part of the rater. OBJECTIVE: To present and evaluate an automated method (AM) to determine the CSP. METHODS: The CSP of 11 healthy controls was recorded using stimulus intensities 20 and 50% above the resting motor threshold (RMT). The mean CSP duration obtained by the two raters using visual inspection and our automated approach were compared. RESULTS: The interclass correlation coefficient (ICC) between the two raters and the AM was 0.99 at 150% of RMT and was 0.97 at 120% of RMT. The level of pre-stimulus EMG amplitude and sampling rate did not affect agreement between the AM and more conventional visually guided methods. CONCLUSIONS: Our study demonstrates that this AM is a simple, objective and reliable approach for CSP determination. SIGNIFICANCE: The CSP is an important neurophysiological measure of cortical inhibition and its determination by our AM provides a more objective and automated approach compared to visually guided methods.

Adult↗

PubMed2002

Analysis of betamethasone in rat plasma using automated solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry. Determination of plasma concentrations in rat following oral and intravenous administration.

A method is described for the determination of betamethasone in rat plasma by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The analyte was recovered from plasma by solid-phase extraction and subsequently analyzed by LC-MS-MS. A Packard Multiprobe II, an automated liquid handling system, was employed for the preparation and extraction of a 96-well plate containing unknown plasma samples, standards and quality control samples in an automated fashion. Prednisolone, a structurally related steroid, was used as an internal standard. Using the described approach, a limit of quantitation of 2 ng/ml was achieved with a 50 microl aliquot of rat plasma. The described level of sensitivity allowed the determination of betamethasone concentrations and subsequent measurement of kinetic parameters of betamethasone in rat. Combination of automated plasma extraction and the sensitivity and selectivity of LC-MS-MS offers a valuable alternative to the methodologies currently used for the quantitation of steroids in biological fluids.

Administration, Oral↗

PubMed2003

A device for automated direct sampling and quantitation from solid-phase sorbent extraction cards by electrospray tandem mass spectrometry.

A new solid-phase extraction (SPE) device in the 96-well format (SPE Card) has been employed for automated off-line sample preparation of low-volume urine samples. On-line automated analyte elution via SPE and direct quantitation by micro ion spray mass spectrometry is reported. This sample preparation device has the format of a microtiter plate and is molded in a plastic frame which houses 96 separate sandwiched 3M Empore sorbents (0.5-mm-thickness, 8-microm particles) covered on both sides by a microfiber support material. Ninety-six discrete SPE zones, each 7 mm in diameter, are imbedded into the sheet in the conventional 9-mm pitch (spacing) of a 96-well microtiter plate. In this study one-quarter of an SPE Card (24 individual zones) was used merely as a convenience. After automated off-line interference elution of applied human urine from 24 samples, a section of SPE Card is mounted vertically on a computer-controlled X, Y, Z positioner in front of a micro ion spray direct sampling tube equipped with a beveled tip. The beveled tip of this needle robotically penetrates each SPE elution zone (sorbent disk) or stationary phase in a serial fashion. The eluted analytes are sequentially transferred directly to a microelectrosprayer to obtain tandem mass spectrometric (MS/MS) analysis. This strategy precludes any HPLC separation and the associated method development. The quantitative determination of Ritalin (methylphenidate) from fortified human urine samples is demonstrated. A trideuterated internal standard of methylphenidate was used to obtain ion current response ratios between the parent drug and the internal standard. Human control urine samples fortified from 6.6 to 3300 ng/mL (normal therapeutic levels have been determined in other studies to be between 50 and 100 ng/mL urine) were analyzed and a linear calibration curve was obtained with a correlation coefficient of 0.9999, where the precision of the quality control (QC) samples ranged from 9.6% at the 24 ng/mL QC level to 1.2% at the 3000 ng/mL QC level, and the accuracy for the four levels of QC samples ranged from 98.1% to 100.3%. The QC samples were prepared at four concentrations which included 24, 240, 1200, and 3000 ng/mL, respectively. The run time per sample in this work was 1.5 min not including the sample preparation time.

Automation↗

PubMed2002

Automated postprocessing of electrospray LC/MS data for profiling protein expression in bacteria.

We describe an integrated approach for automating protein analysis of bacterial cell extracts. The method uses electrospray LC/MS to generate chromatographic profiles of proteins present in an extract, along with a software program that automates the data analysis. The software program, Retana, automates the sequential summing, centroiding, and deconvolution of multiply charged proteins present in consecutive scans of the LC/MS analysis. This procedure generates a concise, single spectrum of proteins present in the extract, along with their retention time and relative abundance. A comparison of the method with "whole cell" MALDI analysis demonstrates improved mass resolution and mass accuracy, along with the appearance of a greater number of proteins. Additionally, it is possible to compare protein expression among strains of bacteria by normalizing the relative abundance of similar proteins in each analysis.

Automation↗

PubMed1999

A molecular scanner to automate proteomic research and to display proteome images.

Identification and characterization of all proteins expressed by a genome in biological samples represent major challenges in proteomics. Today's commonly used high-throughput approaches combine two-dimensional electrophoresis (2-DE) with peptide mass fingerprinting (PMF) analysis. Although automation is often possible, a number of limitations still adversely affect the rate of protein identification and annotation in 2-DE databases: the sequential excision process of pieces of gel containing protein; the enzymatic digestion step; the interpretation of mass spectra (reliability of identifications); and the manual updating of 2-DE databases. We present a highly automated method that generates a fully annoated 2-DE map. Using a parallel process, all proteins of a 2-DE are first simultaneously digested proteolytically and electro-transferred onto a poly(vinylidene difluoride) membrane. The membrane is then directly scanned by MALDI-TOF MS. After automated protein identification from the obtained peptide mass fingerprints using PeptIdent software (http://www.expasy.ch/tools/peptident.html + ++), a fully annotated 2-D map is created on-line. It is a multidimensional representation of a proteome that contains interpreted PMF data in addition to protein identification results. This "MS-imaging" method represents a major step toward the development of a clinical molecular scanner.

Automation↗

PubMed2002

Automated analysis of nanomolar concentrations of phosphate in natural waters with liquid waveguide.

Concentrations of phosphate in natural waters are often below the detection limits of conventional nutrient autoanalyzers, by either gas-segmented continuous-flow analysis orflow injection analysis. A liquid waveguide capillary flow cell has been used to extend the sensitivity of a conventional autoanalyzer for the automated analysis of nanomolar concentrations of phosphate in natural waters. Total reflection of light can be achieved within the liquid core of the flow cell because the refractive index of a cell wall coated with Teflon 1600 is lower than that of water. This property allows the manufacturers to construct long liquid waveguide capillary flow cells in a helical, rather than a linear shape, with compact dimensions. A small sample volume is required because the internal volume of a 2-m long capillary flow cell is only approximately 0.5 cm3. Adaptation of this long flow cell to autoanalyzers significantly enhances the sensitivity of automated colorimetric analysis of phosphate with a molybdenum blue method, allowing for the accurate and precise determination of nanomolar concentrations of phosphate in natural waters. The advantages of this technique are a low detection limit (0.5 nM), a small sample volume (2 mL), high precision (2% at 10 nM levels), and automation for the rapid analysis of a large number of samples.

Automation↗

PubMed2000

Evaluation of a psychophysiologically controlled adaptive automation system, using performance on a tracking task.

Three experiments were conducted to evaluate the performance of a psychophysiologically controlled adaptive automation system. Subjects were asked to perform a compensatory tracking task while their electroencephalogram (EEG) was recorded and an engagement index was derived from the EEG, using the alpha, beta, and theta bandwidths: beta/(alpha + theta) and beta/theta. In Experiment I, EEG was recorded from three different sites: frontal, parietal, and temporal. Although tracking performance did not differ as a function of site, the number of task mode allocations was greater under a negative feedback contingency than under a positive feedback contingency. This effect was seen primarily from frontal sites. Experiments II and III evaluated the adaptive automation system, using extended runs under positive and negative feedback with either a slope (Experiment II) or absolute (Experiment III) criterion used to drive the system. Using either criterion, performance was found to be significantly better under negative feedback. Future evaluation and use of psychophysiologically controlled adaptive automation systems are discussed.

Adaptation, Physiological↗

PubMed2001

SANE (Structure Assisted NOE Evaluation): an automated model-based approach for NOE assignment.

A reliable automated approach for assignment of NOESY spectra would allow more rapid determination of protein structures by NMR. In this paper we describe a semi-automated procedure for complete NOESY assignment (SANE, Structure Assisted NOE Evaluation), coupled to an iterative procedure for NMR structure determination where the user is directly involved. Our method is similar to ARIA [Nilges et al. (1997) J. Mol. Biol., 269, 408-422], but is compatible with the molecular dynamics suites AMBER and DYANA. The method is ideal for systems where an initial model or crystal structure is available, but has also been used successfully for ab initio structure determination. Use of this semi-automated iterative approach assists in the identification of errors in the NOE assignments to short-cut the path to an NMR solution structure.

Automation↗

PubMed2000

Automated immunofluorescence for enumeration of selected taxa in supragingival dental plaque.

The present study investigated a recently developed automated image analysis technique for its applicability to the enumeration of selected bacteria in supragingival dental plaque. Following initial calibration, the system is capable to count fluorescence-labeled target cells in up to 48 samples without user interference. Test samples contained a characteristic mixture of planktonic bacteria, small almost planar bacterial aggregates, and large, virtually indisruptable clumps with cells from multiple species. Due to their complex composition, these samples provided a challenging validation step for the image analysis system. Automated enumeration of target bacteria was compared with visual counting of the fluorescence-labeled bacteria. Results are shown for six taxa (Actinomyces naeslundii, Fusobacterium nucleatum, Prevotella intermedia/Prev. nigrescens, Streptococcus gordonii/Strep. oralis/Strep. sanguis, Strep. sobrinus, and Veillonella dispar/ V. parvula) with characteristic differences in abundance, cell morphology and aggregation behavior. Results revealed good correspondence between the two enumeration techniques (correlation coefficients ranging from 0.77 to 0.92) provided that the portion of target bacteria exceeded 0.05% of the total bacterial cell number. This work demonstrates the applicability and usefulness of fully automated immunofluorescence to analyze such complex ecosystems as supragingival dental plaque.

Actinomyces↗

PubMed2026

DeepLabCut-based automated system reveals diverse temperature tolerance among medaka strains and related Oryzias species.

Temperature is a critical environmental factor influencing the physiology and behavior of ectothermic animals, yet conventional methods for evaluating thermal tolerance in fish rely on subjective manual observation of loss of equilibrium (LOE), limiting experimental throughput and introducing observer bias. Here, we developed an automated temperature tolerance evaluation system integrating DeepLabCut-based pose estimation with custom image processing algorithms to objectively quantify the timing of LOE during thermal stress tests. Our system incorporated region partitioning and color transformation preprocessing to improve keypoint detection accuracy, followed by a classification model combining ResNet34-based frame features with keypoint coordinates to objectively determine the timing of LOE without manual observation. Validation against manual annotation showed that the automated system achieved an accuracy comparable to the natural variability between trained investigators, and outperformed naive human observers, supporting its validity as an objective and reproducible alternative to manual scoring. Using this system, we characterized cold and heat tolerance across six medaka strains (Oryzias latipes: d-rR/TOKYO, HB11A, OK-Cab, HO5 and HdrR-II1; O. sakaizumii: HNI-II). Cold and heat tolerance assessment revealed inter-strain variation, with HdrR-II1 among the most cold- and heat-tolerant strains and HNI-II the least tolerant of both cold and heat stress. We further evaluated cold tolerance in medaka-related species (O. sinensis, O. cabaranensis, O. curvinotus, O. luzonensis, O. celebensis, and O. javanicus) and zebrafish (Danio rerio), revealing substantial interspecific variation that broadly corresponded with latitudinal distribution. O. latipes, distributed at the highest latitudes among the tested species, exhibited the greatest cold tolerance, whereas O. celebensis, O. javanicus, and other tropical or low-latitude species showed comparatively low cold tolerance. Our automated system provides a robust, high-throughput platform for thermal tolerance evaluation and, combined with the genetic and genomic resources available in medaka, establishes a foundation for elucidating the molecular mechanisms underlying temperature adaptation in fish.

Animals↗

PubMed2026

Automated segmentation and length measurement of metacarpal and phalangeal bones for hand radiograph evaluation.

Evaluating hand and wrist radiographs is essential in pediatric endocrinology and clinical genetics, particularly for the assessment of suspected skeletal anomalies. In this study, we present Auto-Bone-Caliper, an automated system for the segmentation and length measurement of metacarpal and phalangeal (M&P) bones, trained and evaluated on public datasets comprising both normal and dysmorphic cases. We first introduce InstanceSAM, a two-stage framework that detects and segments all 19 M&P bones in pediatric hand radiographs, achieving Dice scores of 98.7% for normal bones and 95.0% for dysmorphic bones. We further develop and evaluate three methods for bone-length estimation, identifying a k-means-based approach as the most accurate, with relative errors of 2.2% for normal bones and 4.5% for dysmorphic bones. Our automated pipeline, Auto-Bone-Caliper, integrates InstanceSAM with the k-means-based length-estimation method. To enable scale-independent downstream analyses, we derive relative bone-length measures from the automated measurements. Using these relative measures, we statistically compare measurements obtained using Auto-Bone-Caliper on an independent dataset with a healthy reference catalog of normal bone morphologies, observing a high level of agreement (Wasserstein-1 distance = 0.012). Finally, we demonstrate a potential clinical use case of Auto-Bone-Caliper by obtaining relative metacarpophalangeal pattern profiles for three genetic conditions, namely Turner syndrome, achondroplasia, and pseudohypoparathyroidism. Our results highlight the potential of the Auto-Bone-Caliper to streamline and standardize M&P length measurement, providing an objective and reproducible tool suitable for clinical application.

Humans↗

PubMed2000

Transplantation of CD34+ peripheral blood cells selected using a fully automated immunomagnetic system in patients with high-risk breast cancer: results of a prospective randomized multicenter clinical trial.

Tumor contamination of autologous peripheral blood stem/progenitor cell grafts occurs in a substantial proportion of high-risk breast cancer patients, and the possibility that such contamination may contribute to relapse has focused attention on methods for removal of the contaminating cells prior to transplantation. One such approach is positive selection of CD34+ cells. A fully automated immunomagnetic cell selection system has recently been introduced to facilitate the positive selection process. A multicenter randomized clinical trial was designed to evaluate the capacity of CD34+ cells isolated using the fully automated system to support prompt hematopoietic reconstitution following high-dose chemotherapy in high-risk breast cancer patients, as well as to assess the safety and tolerability of the CD34+ cell transplants. In recipients of isolated CD34+ cells, the median time to an absolute neutrophil count > or =500/microl was 10 days, a value identical to that observed in patients receiving unfractionated apheresis collections. In the isolated CD34+ cell recipients median time to a platelet count > or =20 000/microl was 12 days, compared with 10 days in the unfractionated cell group. There were no statistically significant differences between the groups in median time to neutrophil or platelet engraftment. Infusion of autologous cells was well tolerated by the study groups. There were no inter-group differences in the incidence of infections, need for platelet transfusions, or duration of hospitalization. Isolated CD34+ cells were high in purity and sufficient in number for use in autologous transplantation. The fully automated immunomagnetic cell selection system affords an efficient and time-saving option for isolation of CD34+cells to be used as autologous grafts in high-risk breast cancer patients, and the isolated CD34+ cells support undelayed hematopoietic reconstitution.

Adult↗