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PubMed1997

Automated combined assignment of NOESY spectra and three-dimensional protein structure determination.

A procedure for automated protein structure determination is presented that is based on an iterative procedure during which the NOESY peak list assignment and the structure calculation are performed simultaneously. The input consists of a list of NOESY peak positions and a list of chemical shifts as obtained from sequence-specific resonance assignment. For the present applications of this approach the previously introduced NOAH routine was implemented in the distance geometry program DIANA. As an illustration, experimental 2D and 3D NOESY cross-peak lists of six proteins have been analyzed, for which complete sequence-specific 1H assignments are available for the polypeptide backbone and the amino acid side chains. The automated method assigned 70-90% of all NOESY cross peaks, which is on average 10% less than with the interactive approach, and only between 0.8% and 2.4% of the automatically assigned peaks had a different assignment than in the corresponding manually assigned peak lists. The structures obtained with NOAH/DIANA are in close agreement with those from manually assigned peak lists, and with both approaches the residual constraint violations correspond to high-quality NMR structure determinations. Systematic comparisons of the bundles of conformers that represent corresponding automatically and interactively determined structures document the absence of significant bias in either approach, indicating that an important step has been made towards automation of structure determination from NMR spectra.

Magnetic Resonance Spectroscopy↗
PubMed2002

Automated assignment of NOESY NMR spectra using a knowledge based method (KNOWNOE).

Automated assignment of NOESY spectra is a prerequisite for automated structure determination of biological macromolecules. With the program KNOWNOE we present a novel, knowledge based approach to this problem. KNOWNOE is devised to work directly with the experimental spectra without interference of an expert. Besides making use of routines already implemented in AUREMOL, it contains as a central part a knowledge driven Bayesian algorithm for solving ambiguities in the NOE assignments. These ambiguities mainly arise from chemical shift degeneration which allows multiple assignments of cross peaks. Using a set of 326 protein NMR structures, statistical tables in the form of atom-pairwise volume probability distributions (VPDs) were derived. VPDs for all assignment possibilities relevant to the assignments of interproton NOEs were calculated. With these data for a given cross peak with N possible assignments Ai (i = 1,...,N) the conditional probabilities P(Ai, a/V0) can be calculated that the assignment Ai determines essentially all (a-times) of the cross peak volume V0. An assignment Ak with a probability P(Ak, a/V0) higher than 0.8 is transiently considered as unambiguously assigned. With a list of unambiguously assigned peaks a set of structures is calculated. These structures are used as input for a next cycle of iteration where a distance threshold Dmax is dynamically reduced. The program KNOWNOE was tested on NOESY spectra of a medium size protein, the cold shock protein (TmCsp) from Thermotoga maritima. The results show that a high quality structure of this protein can be obtained by automated assignment of NOESY spectra which is at least as good as the structure obtained from manual data evaluation.

Algorithms↗
PubMed2004

Rapid determination of bovine lactoferrin in dairy products by an automated quantitative agglutination assay based on latex beads coated with F(ab')2 fragments.

UNLABELLED: This study evaluated an automated immunoassay for bovine lactoferrin (LF) in dairy products based on latex beads coated with F(ab')2 fragments. METHODS: F(ab')2 fragments were obtained by pepsin digestion of rabbit anti-bovine LF (IgG fraction) and polystyrene latex beads were coated with the F(ab')2 fragments. We used the beads to develop a rapid and homogeneous light scatter immunoassay employing an autoanalyzer (the Automated Latex assay). The Automated Latex assay was easy to perform and could rapidly determine bovine lactoferrin in lactoferrin-supplemented products. It was sensitive enough for testing products and showed good precision.

Animals↗
PubMed2004

Assessment of left ventricular volume and function by integration of simplified 3D echocardiography, tissue harmonic imaging and automated extraction of endocardial borders.

BACKGROUND: Quantitative three-dimensional echocardiography (3DEcho) of left ventricle (LV) is still limited because of the need for manually tracing endocardial borders: this can increase observer variability depending on the quality of fundamental (FUND) 2D images. We tested the combination of a simplified 3DEcho technique for LV reconstruction with automated endocardial border detection (Digital Echo Quantification, DEQ) and tissue harmonic imaging (THI) for enhancement of endocardium-cavity interface. METHODS: Twenty-five consecutive patients with ischaemic heart disease and dilated or distorted LV underwent 3DEcho and gated-SPECT nuclear examinations evaluating: (a) end-diastolic and end-systolic volumes (EDV, ESV); (b) ejection fraction (EF); (c) volume/time curve (VTC). Thereafter, 3DEcho was applied to 47 patients with acute myocardial infarction (AMI) at pre-discharge and 6 months to evaluate remodelling. RESULTS: Integrated 3DEcho values in THI modality were obtained in 84% of patients and correlated well with nuclear data for EDV (r = 0.95, mean difference = -2.2 +/- 15.8 ml), ESV (r = 0.98, mean difference = -3.5 +/- 10.2 ml), and EF (r = 0.82, mean difference = 0.6 +/- 6.4%; all mean differences NS vs. 0), with an interobserver variability of 4.9, 5.7 and 8.2% for EDV, ESV and EF respectively. Automated VTC by 3DEcho in THI modality reproduced well that obtained by nuclear technique (r = 0.96) and allowed recognition of LV remodelling in 36% of patients at 6 months. Integrated 3DEcho values in FUND modality were obtained only in 52% of patients and showed much higher errors and interobserver variability. CONCLUSIONS: THI permits accurate 3D reconstruction of LV borders detected by DEQ, allowing automated VTC throughout the cardiac cycle as well as study of LV remodelling.

Algorithms↗
PubMed2001

HLA-DRB fluorotyping by dark quenching and automated analysis.

In fluorescence-based sequence-specific primed polymerase chain reaction (PCR), referred to as fluorotyping for HLA typing, a major problem is the overlap of the emission spectra of the single dyes. In order to increase the robustness of the previously described HLA-DRB1,3,4,5 low-resolution fluorotyping method, we have constructed two probes quenched by the non-fluorescent acceptor dye DABCYL. The HLA-DRB-specific probe was labeled with FAM, and the internal control probe with TAMRA, respectively, as reporter fluorescent dyes. TAMRA was replaced by DABCYL as a quencher, which led to increased robustness and better discrimination between negative and positive amplification results. ROX was used as a reference to normalize the fluorescence of the reporter dyes. Moreover, as FAM and TAMRA differ strongly by their emission maxima, HLA-DRB-specific and internal control amplification could be clearly distinguished. To further automate data analysis, the software of the TaqMan system 7700 was supplemented by an EXCEL-based calculation table, which directly took over the data. Using modified fluorotyping chemistry and automated data analysis, a total of 201 DNA samples was typed correctly. In summary, HLA-DRB fluorotyping by dark quenching and automated analysis proved to be a robust and reliable tool for research and routine purposes.

Electronic Data Processing↗
PubMed1997

A fully automated comparative microaneurysm digital detection system.

A fully automated digital image processing system, which provides an objective and repeatable way to quantify microaneurysms in digitised fluorescein angiograms, has been developed. The automated computer processing includes registration of same-eye retinal images for serial studies, cutting of regions-of-interest centred on the fovea, the detection of microaneurysms and the comparison of serial images for microaneurysm turnover. The microaneurysm detector was trained against a database of 68 images of patients with diabetes containing 394 true microaneurysms, as identified by an ophthalmologist. The microaneurysm detector achieved 82% sensitivity with 2.0 false-positives per image. An independent test set, comprising 20 images containing 297 true microaneurysms, was used to compare the microaneurysm detector with clinicians. The microaneurysm detector achieved a sensitivity of 82% for 5.7 false-positives per image, whereas the clinician receiver-operator-characteristic (ROC) curve gives 3.2 false-positives per image at a sensitivity of 82%. It is concluded that the computer system can reliably detect microaneurysms. The advantages of the computer system include objectivity, repeatability, speed and full automation.

Aneurysm↗
PubMed1989

Automated spinal cord monitoring for spinal surgery.

A microcomputer-based automated spinal cord monitoring system was developed and 56 spinal operations have been carried out with it since 1986. The system comprises an electrodiagnostic system (Dantec 2000M), an FM tape recorder, a channel selector, an electrical relay, a Modem and a microcomputer which controls all of the elements. The program allowed for automated recording and analysis of evoked potentials during spinal surgery and alarm signals were generated according to the criteria of amplitude decrease and latency delay. It was demonstrated that automated spinal cord monitoring in the operating room was performed easily, reliably and safely allowing the surgeon to react quickly to possible compromise of the spinal cord. The design and usefulness of the system is described in this report.

Diagnosis, Computer-Assisted↗
PubMed1999

Enhanced assessment of allogeneic bone marrow transplant engraftment using automated fluorescent-based typing.

Traditional qualitative gel electrophoresis approaches lack accurate' and quantitative assessment of mixed chimerism in BMT patients. The likelihood of informative markers is greatly increased using simultaneous amplification of 10 highly polymorphic loci with fluorescent-labeled primers in an automated DNA sequencer. This allows for more precise interpretation of mixed chimerism with a detection level approximating 1%. To evaluate this approach to quantitative assessment of chimeric populations we mixed varying proportions of samples from two unrelated donors, by either mixing aliquots of DNA isolated from whole blood, or by first counting the white blood cells and mixing varying proportions of cells together prior to DNA isolation. The allelic-peak area ratios were identical to allelic-peak height ratios and corresponded to the proportion of mixed DNA, regardless of the method used to create the mixture. Formulas to provide routine, consistent and quantitative interpretation of mixed chimerism are presented. We analyzed 14 allograft recipients and one autologous BMT patient with transfusion-induced GVHD. In all cases, at least four out of nine markers were informative. Inter-laboratory concordance of results was also obtained with an eight marker panel using an automated Alf-Express. In conclusion, the automated DNA fluorescent-labeled primer approach using an eight to 10 marker panel is quantitative and informative in assessing chimerism.

Alleles↗
PubMed1997

Automated high resolution PCR fragment analysis for identification of clonally rearranged immunoglobulin heavy chain genes.

The development of rapid polymerase chain reaction (PCR) protocols for amplification of rearranged heavy chain immunoglobulin (IgH) gene sequences has facilitated the identification of clonal IgH rearrangements in non-Hodgkin's lymphomas (NHL) and leukemias of B lineage. In the present report we have explored the recently described improved strategy for assessment of clonality of rearranged immunoglobulin heavy chain (IgH) genes in more detail in a series of 101 B cell malignancies and 50 polyclonal controls. The assay is based on an IgH-PCR with an automated fluorescence-based strategy for PCR detection of IgH gene rearrangements. Third complementarity determining region (IgH-CDR3) sequences were amplified using fluorescent dye labeled consensus primers homologous to the corresponding variable (V[H]) and joining (J[H]) gene segments in combination with a thermostable proofreading DNA polymerase. PCR products were size separated on a high resolution polyacrylamide gel and analyzed for clonality by exact size determination and fluorescence quantification in an automated DNA sequencer. PCR findings obtained with the optimized IgH-CDR3-PCR assay showed an overall monoclonality detection rate of 97% (97 of 101 cases with B cell neoplasms). The specificity was 100% as determined by analysis of 50 controls, all of which gave polyclonal PCR results. We found a high rate of monoclonal IgH-CDR3-PCR results not only in the leukemias and diffuse lymphoma but also in the group of follicular lymphoma, where a high rate of false negative results is frequently reported in the literature. In summary, we identified monoclonal IgH-CDR3 junctions in 55 out of 59 cases (93%) with B cell lymphoma and in 42 of 42 (100%) cases with leukemia, immunocytoma and multiple myeloma. The results demonstrate that automated fluorescence detection of IgH-CDR3-PCR products is an ideal tool for detection of clonal and polyclonal lymphoid B cells. In combination with allele-specific primers the procedure may improve current experimental approaches to detect occult malginant B cells during initial staging and follow-up of NHL and ALL patients.

Alleles↗
PubMed1998

Development of an automated measurement system using a diffusion scrubber and high-performance liquid chromatography for the monitoring of formaldehyde and acetaldehyde in automotive exhaust gas.

An automated measurement system for monitoring formaldehyde (HCHO) and acetaldehyde (CH3CHO) in automotive exhaust gas by using a diffusion scrubber in combination with high-performance liquid chromatography (HPLC) was developed. HCHO and CH3CHO are effectively collected by the diffusion scrubber, which consists of a hydrophobic porous PTFE tube disposed concentrically within a Pyrex-glass tube and a scrubbing solution. 2,4-Dinitrophenylhydrazine is used as the scrubbing solution for trapping HCHO and CH3CHO, which are derivatized to formaldehyde 2,4-dinitrophenylhydrazone (DNPH-HCHO) and acetaldehyde 2,4-dinitrophenylhydrazone (DNPH-CH3CHO), respectively, with phosphoric acid as an acid catalyst. After the collection of the gas sample, the sample solution in the diffusion scrubber is injected into the HPLC system and DNPH-HCHO and DNPH-CH3CHO are separated and determined. All measurement operations are sequenced by a programmable controller and an automated continuous measurement can be performed at 10 min intervals. The collection efficiencies of HCHO and CH3CHO were higher than 97% at a gas flow rate of 0.21 min-1. The detection limit (3 sigma of the blank value) was 0.001 ppm v/v for HCHO and CH3CHO for a 1.61 gas sample volume. No interference of co-existing nitrogen dioxide (NO2) in the collection of HCHO and CH3CHO was observed. The average concentration of HCHO in the exhaust gas from methanol-fueled vehicles was 77.3 ppm v/v (n = 5) in the cold-phase mode when engines were first started. In the hot-phase mode, the average concentration of HCHO was 3.3 ppm v/v (n = 15). The concentrations of HCHO measured by this automated measurement system were in good agreement with those obtained using the impinger-HPLC method.

Acetaldehyde↗
PubMed2001

Automated measurement system for H2O2 in the atmosphere by diffusion scrubber sampling and HPLC analysis of Ti(IV)-PAR-H2O2 complex.

An automated selective measurement system for monitoring hydrogen peroxide (H2O2) in the atmosphere was developed (using a diffusion scrubber coupled to a high performance liquid chromatography) due to the importance of H2O2 in understanding tropospheric chemistry and its harmful effects on vegetation. H2O2 in the atmosphere was effectively collected by a diffusion scrubber, which consisted of a hydrophobic porous polytetrafluorethylene (PTFE) tube positioned concentrically within a Pyrex glass tube. Titanium(IV)-4-(2-pyridylazo)resorcinol (Ti(IV)-PAR) solution acidified at pH 2.2 was used as the scrubbing solution for the diffusion scrubber. After the collection of the air sample by the diffusion scrubber, the pH value of the Ti(IV)-PAR scrubbing solution was adjusted to pH 11.9 to form a stable complex of Ti(IV)-PAR-H2O2. An aliquot of the sample solution was injected into a high performance liquid chromatograph equipped with a semi-micro-reversed-phase column and a spectrophotometric detector set at 508 nm for separating and determining the Ti(IV)-PAR-H2O2 complex. The automated measurement could be performed at 60 min intervals. The collection efficiency of H2O2 was higher than 98% at an air flow rate of 1.0 l min-1. The detection limit (3 sigma of the blank value) of H2O2 was 9 parts per trillion by volume (pptv) for an air sampling volume of 55.1. The interference from coexisting O3 or SO2 in the atmosphere was negligible during the collection of H2O2 by the diffusion scrubber. The developed automated measurement system was suitable for monitoring H2O2 in the atmosphere.

Air Pollutants↗
PubMed2004

Automated measurement and calibration of reactive volatile halogenated organic compounds in the atmosphere.

The automated calibration and analysis of very low mixing ratios of the reactive volatile organic halocarbons CH(3)I, CHCl(3), C(2)H(5)I, 2-C(3)H(7)I, CH(2)Br(2), CH(2)ClI, CHBr(2)Cl, 1-C(3)H(7)I, CH(2)BrI, CHBr(3) and CH(2)I(2) for long term atmospheric field measurements are described. Analytes were pre-concentrated from 3 l of air onto an adsorbent trap cooled to -10 [degree]C using Peltier plates, and rapidly transferred to a gas chromatograph (GC) by resistive heating. A two stage Carboxen 1016/Carbotrap C adsorbent trap allowed good analyte recovery and rapid desorption without the need for post-desorption cryofocussing. Halocarbons were detected using a mass spectrometer (MS) in selective ion mode. Detection limits were between 0.02 and 0.12 pptv (parts per trillion by volume) for approximately hourly samples of CHCl(3), CH(3)I, C(2)H(5)I, 1-C(3)H(7)I, 2-C(3)H(7)I, CH(2)ClI, CH(2)Br(2), CHBr(2)Cl, CH(2)BrI, CHBr(3) and CH(2)I(2) with a precision of 3-8%. A novel calibration system was constructed which utilised fixed volume (50 [micro sign]l) injections of the output of thermostatted permeation tubes into a stream of nitrogen gas in order to dilute parts per million by volume (ppmv) mixing ratios into pptv. The calibration was completely automated, allowing multi-point calibrations during routine operation. The overall accuracy of the measurements is estimated to be +/-15%. The instrument was used continuously for automated atmospheric measurements during a 4-month research cruise from Germany to Antarctica, and a 6 week field campaign at Mace Head, Ireland. The results for CHCl(3) during the latter campaign were within 13% of measurements made by a GC-MS operating continuously at the site within the long term Advanced Global Atmospherics Gases Experiment.

Air Pollutants↗
PubMed1999

Automated grammatical tagging of child language samples.

Recent studies of the automated grammatical categorization ("tagging") of words using probabilistic methods have reported substantial levels of accuracy-over 95% agreement with manual tagging for words from a variety of texts. However, the texts with which this method has been tested were written by adults and edited by publishers. The present study examined the accuracy with which such methods could tag transcribed conversational language samples from 30 normally developing children. On a word-by-word basis, automated accuracy levels ranged from 92.9% to 97.4%, averaging 95.1%. Accuracy at correctly tagging whole utterances was lower, ranging from 60.5% to 90.3%, with an average of 77.7%. Probabilistic methods of coding language samples hold potential as a viable tool for child language research. Further study and improvement of automated grammatical tagging is warranted and necessary before widespread use can be made of this technology.

Child↗
PubMed1997

Automated reticulocyte counting: evaluation of the Coulter STKS Haematology Analyser reticulocyte counting function.

This study evaluated reticulocyte counting with the automated reticulocyte function of the Coulter STKS Haematology Analyser. This is an upgrade option for Coulter STKS and MAXIM haematology analysers. Reticulocyte counts obtained with the automated reticulocyte counting function were compared with those obtained by visual counting. Reticulocyte counting with both methods gave excellent comparability with a correlation coefficient of 0.98. Results were consistent with the well documented imprecision of the manual method with a coefficient of variation (CV) of 16-22%. In contrast, the automated reticulocyte counting function was more precise with a CV of 12.3%. In both cases, counts were stable after storage for 24 h at room temperature and 4 degrees C. Our results suggest that the use of this upgrade will be beneficial for many laboratories.

Autoanalysis↗
PubMed1997

Evaluation of the Abbott Cell Dyn 4000 automated fluorescent reticulocyte measurements: comparison with manual, FACScan and Sysmex R1000 methods.

The Abbott Cell Dyn 4000 (CD4000) is the first haematology analyser in which fully-automated reticulocyte measurements can be routinely determined by fluorescence as part of the full blood count. This communication reports the first evaluation of this method which was undertaken by three independent reference laboratories in Belgium, Germany and Italy. A total of 695 different samples was entered into the study which was designed to compare CD4000 reticulocyte information (enumeration and qualitative maturational data) with results determined in parallel with the existing manual (supravital staining) reference procedure, and two semi-automated fluorescent assays (Becton Dickinson FACScan and Sysmex R1000 instruments). These studies revealed good agreement between the CD4000 and the manual procedure, with no inter-method bias. Comparison of CD4000 and FACScan reticulocyte measurements, however, indicated a distinct tendency for the FACScan to give higher reticulocyte estimates than the CD4000. Finally, the comparison of the CD4000 with the Sysmex R1000 showed excellent agreement in the range 0-6% reticulocytes, although there was some inter-method bias in the higher range (> 15%). Analysis of agreement levels between the methods using specific 'clinical decision points' confirmed the tendency for overestimation by the FACScan, in that 58% of the samples with a reticulocytopenia of < 0.5% as defined by the CD4000 gave FACScan results within the normal range (0.5-1.8%). In contrast, there was absolute agreement between the CD4000 and the Sysmex R1000 for all reticulocytopenias. Comparison (195 samples) of instrument fluorescent reticulocyte maturation profiles demonstrated an exponential relationship (r = 0.78) between CD4000 IRF and R1000 HFR (highly fluorescent reticulocyte fraction) values. The suggestion that the CD4000 IRF values includes some of the MFR as well the HFR reticulocyte fraction was confirmed as the correlation between the CD4000 IRF and the Sysmex R1000 MFR plus HFR percentages was linear (r = 0.82). This study confirms a high performance level for the CD4000 automated fluorescent reticulocyte method.

Autoanalysis↗
PubMed2001

Automated ribotyping and random amplified polymorphic DNA analysis for molecular typing of Salmonella enteritidis and Salmonella typhimurium strains isolated in Italy.

AIMS: The ability of automated ribotyping and random amplified polymorphic DNA (RAPD) analysis to differentiate Salmonella enteritidis and Salmonella typhimurium isolates in relation to their origin was evaluated. METHODS AND RESULTS: The restriction enzymes EcoRI, PvuII and PstI, and the random primers OPB17 and P1254, were tested for ribotyping and RAPD analysis, respectively. Seventeen subtypes were identified among the isolates of the two pathogenic Salmonella serovars using the RiboPrinter, and 25 subtypes using RAPD. CONCLUSIONS: The greatest degree of genetic diversity was observed among Salm. typhimurium isolates using both automated ribotyping (Simpson's index of discrimination 0878) and RAPD (Simpson's index of discrimination 0886). SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results of this research, automated ribotyping and RAPD are two useful genotyping techniques for identifying unique and common subtypes associated with a specific source and location, and provide powerful tools for epidemiological investigations.

Animals↗
PubMed2003

Correction of autofocusing errors due to specimen tilt for automated electron tomography.

Transmission electron microscopy images acquired under tilted-beam conditions experience an image shift as a function of defocus settings - a fact that is exploited as a method for defocus determination in most of the automated tomography data collection systems. Although the method was shown to be highly accurate for a large variety of specimens, we point out that in its original design it can strictly only be applied to images of untilted samples. The application to tilted samples and thus in automated electron tomography is impaired mainly due to a defocus change across the images, resulting in reduced accuracy. In this communication we present a method that can be used to improve the accuracy of the basic autofocusing procedures currently used in systems for automated electron tomography.

Animals↗
PubMed2003

Automated three-dimensional analysis of particle measurements using an optical profilometer and image analysis software.

The automated collection of topographic images from an optical profilometer coupled with existing image analysis software offers the unique ability to quantify three-dimensional particle morphology. Optional software available with most optical profilers permits automated collection of adjacent topographic images of particles dispersed onto a suitable substrate. Particles are recognized in the image as a set of continuous pixels with grey-level values above the grey level assigned to the substrate, whereas particle height or thickness is represented in the numerical differences between these grey levels. These images are loaded into remote image analysis software where macros automate image processing, and then distinguish particles for feature analysis, including standard two-dimensional measurements (e.g. projected area, length, width, aspect ratios) and third-dimensional measurements (e.g. maximum height, mean height). Feature measurements from each calibrated image are automatically added to cumulative databases and exported to a commercial spreadsheet or statistical program for further data processing and presentation. An example is given that demonstrates the superiority of quantitative three-dimensional measurements by optical profilometry and image analysis in comparison with conventional two-dimensional measurements for the characterization of pharmaceutical powders with plate-like particles.

Glass↗