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PubMed1998

[The influence of several factors on automated DNA sequencing].

OBJECTIVE: To investigate the influence of several factors on isothermal automated DNA sequencing. METHODS: The effects of DNA purity and DNA concentration, as well as the quality of water used during sequencing on automated DNA sequencing results were retrospectively analyzed. RESULTS: When the DNA was not pure enough, the nucleotide curve showed lower ratio of signal to noise, or compression would occur, leading to difficulty in correctly judging the nucleotides; the readable length of nucleotides was shorter when low concentration of DNA was used; when the quality of water used in sequencing reaction and polyacrylamide gel solution was not high enough, the peaks of nucleotides became wider and flatter, or double peaks would occur, resulting in unreadable sequences. CONCLUSION: DNA purity and concentration as well as quality of water used in sequencing reaction and polyacrylamide gel solution had apparent influence on isothermal automated DNA sequencing. This suggests that high purity of DNA and high quality of water should be used in isothermal automated DNA sequencing; if longer DNA sequence needs to be obtained, DNA concentration should not be too low.

DNA↗
PubMed1998

Automated histometry in quantitative prostate pathology.

OBJECTIVE: To review progress on the development of machine vision and image understanding in prostate tissue histology and to discuss the problems and opportunities afforded to pathology through the use of these techniques. STUDY DESIGN: A variety of concepts in machine vision are explored, and methodologies are described that have been developed to deal with the complexities of histologic imagery. The theory of human vision and its impact on machine vision are discussed. Software has been specifically developed for the analysis of prostate histology, allowing accurate gland segmentation, basal cell identification and measurement of vascularization within lesions. RESULTS: Image interpretation can be achieved using knowledge-based image analysis and the application of local object-oriented processing. This successfully allows an automated quantitative analysis of histologic morphology in the diagnosis of prostate intraepithelial neoplasia and invasive prostatic cancer. The use of low-power image scanning, based on textural or n-gram mapping, permits the development of fully automated devices for the rapid detection of tissue abnormalities. High-power, knowledge-guided scene segmentation can be carried out for the quantitative analysis of cellular features and the objective grading of the lesion. CONCLUSION: Automated tissue section scanning and image interpretation is now possible and holds much promise in prostate pathology and other diagnostically demanding areas. Issues of standardization still need to be addressed, but the development of such systems will undoubtedly enhance our diagnostic capabilities through the automation of time-consuming procedures and the quantitative evaluation of disease processes.

Humans↗
PubMed1998

A randomized controlled trial of automated term composition.

OBJECTIVE: To compare the ability of an Automated Term Composition (ATC) algorithm with non-compositional mappings to provide coverage (exact mappings to a controlled vocabulary) for a randomly selected set of free text entries which were entered as headings to the Impression section of the clinical notes system at the Mayo Foundation. We also compare the results of four evaluators to determine the inter-observer variability and the variance between term sets, with respect to the accuracy of the mappings and the reliability of the failure analysis. METHODS: From a corpus of approximately 1,000,000 unique terms entered into the Impression/Report/Plan section of the clinical notes system in the calendar year 1997, we randomly selected 1,000 terms. We then further randomized these 1,000 terms into two groups of 500 (Sets A and B). We constructed two copies of the same term matching interface, one without ATC (alpha) and one with ATC (beta). We took four expert Indexers and assigned them to one of the following tasks. The first reviewer (R1) compared set A using the alpha program and then set B using the beta program (R1(Aalpha + Bbeta)). The second compared set A using the alpha program and then set B using the alpha program (R2(A + B) alpha). The third compared set B using the beta program and then set A using the beta program (R3(B + A) beta). The fourth compared set A using the beta program and then set B using the alpha program (R4(Abeta + Balpha)). RESULTS: The program with Automated Term Composition mapped 540 out of the 1,000 Concepts correctly (54.0%). The same program without ATC mapped only 276 out of the 1,000 Concepts correctly (27.6%). Therefore the program with ATC was significantly more effective at matching concepts in our problem lists than the same search engine without ATC (p < 0.0001; McNemar Method). These figures result from the comparison of the alpha program with the beta program by reviewers one and four. Failure analysis showed that with the alpha version 425 out of the 724 mismatches were because a base concept was missing from the retrieval set (58.7%) and 299 mismatches were from missing qualifiers or modifiers or both (41.3%). In the beta version of the program (with ATC) 340 out of the 460 mismatches were secondary to there being a missing base concept in the retrieval set (73.9%) and only 120 mismatches due to missing modifiers and or qualifiers (26.1%). CONCLUSIONS: Automated term composition provided significantly better coverage of a randomly chosen set of patient problems, diagnosed at the Mayo Clinic during the 1997 calendar year, when compared with the same information retrieval system without ATC. We believe that these results speak further to the excellent content coverage provided by the UMLS metathesaurus. These authors believe that increased structure, normalization of UMLS content and semantics, and better tools to make use of the currently available content such as automated term composition, are what is needed to leverage the production of commercially viable tools that provide access to controlled vocabularies for medicine.

Abstracting and Indexing↗
PubMed1996

Tissue disaggregation for flow cytometric DNA analysis: comparison of fine-needle aspiration and an automated mechanical procedure.

Flow cytometric DNA analysis was performed on unfixed frozen samples from 56 breast cancer patients. From each patient, two samples were analyzed. The only difference in the handling of the paired samples was the mechanical disaggregation of one sample by fine-needle aspiration compared to an automated mechanical disaggregation method (Medimachine) of the other sample. With the two methods for tissue disaggregation, the same resolution of the DNA histograms was obtained, indicated by median coefficients of variation (CV) of 1.5% for the DNA diploid G1 peaks. Also, the frequencies of DNA diploid and aneuploid cases as well as the fractions of DNA aneuploid cells were comparable. This indicates that the two methods did not differ in ability to detect DNA aneuploid tumor clones. Automated mechanical disaggregation resulted in DNA histograms with significantly less debris and with lower S-phase fractions. In practice, the procedure of automated mechanical disaggregation was rapid, easy, and safer because of minimal handling of the unfixed tissue compared to the fine-needle aspiration.

Automation↗
PubMed1996

Sample preparation and in situ hybridization techniques for automated molecular cytogenetic analysis of white blood cells.

With the advent of in situ hybridization techniques for the analysis of chromosome copy number or structure in interphase cells, the diagnostic and prognostic potential of cytogenetics has been augmented considerably. In theory, the strategies for detection of cytogenetically aberrant cells by in situ hybridization are simple and straightforward. In practice, however, they are fallible, because false classification of hybridization spot number or patterns occurs. When a decision has to be made on molecular cytogenetic normalcy or abnormalcy of a cell sample, the problem of false classification becomes particularly prominent if the fraction of aberrant cells is relatively small. In such mosaic situations, often > 200 cells have to be evaluated to reach a statistical sound figure. The manual enumeration of in situ hybridization spots in many cells in many patient samples is tedious. Assistance in the evaluation process by automation of microscope functions and image analysis techniques is, therefore, strongly indicated. Next to research and development of microscope hardware, camera technology, and image analysis, the optimization of the specimen for the (semi)automated microscopic analysis is essential, since factors such as cell density, thickness, and overlap have dramatic influences on the speed and complexity of the analysis process. Here we describe experiments that have led to a protocol for blood cell specimen that results in microscope preparations that are well suited for automated molecular cytogenetic analysis.

Acetic Acid↗
PubMed1996

Automation of spot counting in interphase cytogenetics using brightfield microscopy.

In situ hybridization techniques allow the enumeration of chromosomal abnormalities and form a great potential for many clinical applications. Although the use of fluorescent labels is preferable regarding sensitivity and colormultiplicity, chromogenic labels can provide an excellent alternative in relatively simple situations, e.g., where it is sufficient to use a centromere specific probe to detect abnormalities of one specific chromosome. When the frequency of chromosomal aberrations is low, several hundreds or even thousands of cells have to be evaluated to achieve sufficient statistical confidence. Since manual counting is tedious, fatiguing, and time consuming, automation can assist to process the slides more efficiently. Therefore, a system has been developed for automated spot counting using brightfield microscopy. This paper addresses both the hardware system aspects and the software image analysis algorithms for nuclei and spot detection. As a result of the automated slide analysis the system provides the frequency spot distribution of the selected cells. The automatic classification can, however, be overruled by human interaction, since each individual cell is stored in a gallery and can be relocated for visual inspection. With this system a thousand cells can be automatically analyzed in approximately 10 min, while an extra 5-10 min is necessary for visual evaluation. The performance of the system was analyzed using a model system for trisomy consisting of a mixture of male and female lymphocytes hybridized with probes for chromosomes 7 and Y. The sensitivity for trisomy detection in the seeding experiment was such that a frequency of 3% trisomic cells could be picked up automatically as being abnormal according to the multiple proportion test, while trisomy as low as 1.5% could be detected after interaction.

Algorithms↗
PubMed2000

Automated assay for nicotinamide adenine dinucleotide (NAD+).

Sulfur mustard (HD), a vesicating chemical warfare compound, has been shown to deplete the nicotinamide adenine dinucleotide (NAD+) content in several cell systems and tissues. This NAD+ depletion has been proposed as an indicator of HD exposure and can be used to evaluate potential antivesicant compounds. To examine NAD+ levels, an automated method based on the alcohol dehydrogenase cycling assay of Jacobson and Jacobson and utilizing a Cobas FARA clinical analyzer has been developed. Automation of this assay led to smaller sample volumes and more efficient use of personnel and resources. The usefulness of this automated method was tested by evaluating the protection, if any, by the topical application of vitamin D or betamethasone against HD-induced NAD+ depletion in skin punches from the hairless guinea pig. The results showed that the samples exposed to HD exhibited significant decreases in NAD+ levels when compared with controls. However, neither vitamin D nor betamethasone demonstrated protection against HD-induced NAD+ depletion. In fact, betamethasone exacerbated the NAD+ depletion when compared with the HD exposed group. This assay appears to be useful for testing potential antivesicant compounds using both in vivo and in vitro exposure systems.

Animals↗
PubMed2002

Automation for continuous analysis on microchip electrophoresis using flow-through sampling.

Automation of electrophoretic microchips for sequential analysis of different samples is demonstrated. This system used an autosampler, which was on-line connected to the microchip and the whole process including sample loading and injection, analysis and data acquisition as well as washing were all automated. Rhodamin B at different concentrations was first loaded into a hydrodynamic flow stream by an autosampler, delivered to the microchip, and then sequentially injected into the electrophoretic microchannel for analysis and detection. Automation was achieved by running two independent programs, one for sample loading by an autosampler and the other one for electrophoretic injection by voltage switching, on the same computer. Using this sampling chip, each loaded volume (0.2-1 microL) can be injected for dozens of electrophoretic analyses (1-10 nL for each injection). The variances caused by the external connections, which did not affect the electrophoretic analysis but would cause band broadening of the loaded sample in the hydrodynamic flow stream, were theoretically deduced. Results indicate that the dead volume (approximately 300 nL) due to the connection fitting on the chip could lead to dilution of the loaded sample by a factor of one when 0.2 microL of sample was loaded. Such a design allows sequential analysis of a series of samples while the running buffer is continuously pumped into the connection capillary as well as microchannels for washing between two loaded samples to minimize cross contamination without human intervention. Using this sampling chip, the required sample amount and handling time can be greatly reduced compared to the manual method.

Automation↗
PubMed1995

Automated linkage analysis in psychiatric disorders.

A genome-wide search for linkage of microsatellite markers to chromosomal loci containing genes responsible for the major psychoses is a laborious task which can be carried out with greater speed and economy by introducing automation to several steps in the procedure. We describe the use of the Automated Linkage Preprocessor (ALP) program for the computer analysis of the waveform generated by fluorescein-labelled markers after electrophoretic separation. (To obtain a copy send a request to A.F. Brown at the below MRC address or use Anonymous FTP to ftp.hgu.mrc.ac.uk. Software is in directory pub/ALP). The program runs on a PC in the Microsoft Windows environment, and is used in conjunction with an automated laser fluorescence (ALF) sequencer (Pharmacia) and its Fragment Manager software to detect and size the PCR products, filter out peaks of fluorescence due to nonallele fragments, and generate genotypes in a format suitable for direct input to standard linkage analysis programs. The method should offer the advantages of speed, accuracy, and reduced cost. Its use in linkage studies in a large family with manic-depressive illness is discussed.

Automation↗
PubMed2005

Automated seizure abatement in humans using electrical stimulation.

The need for novel, efficacious, antiseizure therapies is widely acknowledged. This study investigates in humans the feasibility, safety, and efficacy of high-frequency electrical stimulation (HFES; 100-500 Hz) triggered by automated seizure detections. Eight patients were enrolled in this study, which consisted of a control and an experimental phase. HFES was delivered directly to the epileptogenic zone (local closed-loop) in four patients and indirectly, through anterior thalami (remote closed-loop), to the other four patients for every other automated seizure detection made by a validated algorithm. Interphase (control vs experimental phase) and intraphase (stimulated vs nonstimulated) comparisons of clinical seizure rate and relative severity (clinical and electrographic) were performed, and differences were assessed using effect size. Patients were deemed "responders" if seizure rate was reduced by at least 50%; the remaining patients were deemed "nonresponders." All patients completed the study; rescue medications were not required. There were 1,491 HFESs (0.2% triggered after-discharges). Mean change in seizure rate in the local closed-loop group was -55.5% (-100 to +36.8%); three of four responders had a mean change of -86% (-100 to -58.8%). In the remote closed-loop, the mean change of seizure rate was -40.8% (-72.9 to +1.4%); two of four responders had a mean change of -74.3% (-75.6 to -72.9%). Mean effect size was zero in the local closed-loop (responders: beneficial and medium to large in magnitude) and negligible in the remote closed-loop group (responders: beneficial and medium to large). HFES effects on epileptogenic tissue were immediate and also outlasted the stimulation period. This study demonstrates the feasibility and short-term safety of automated HFES for seizure blockage, and also raises the possibility that it may be beneficial in pharmaco-resistant epilepsies.

Adolescent↗
PubMed1983

Old and new models for office automation.

The emerging generation of office automation systems combines new and existing software and procedures. While managers may be able to select from a broad array of software tools, they may also be required to use certain others. This article discusses organization design as the context for office automation; mature computer-based systems as one application of organization design variables; and emerging office automation systems as another application of those variables. The article concludes that Management Information System models developed for mature systems may be helpful where the use of software application is required for the individual worker; diffusion of innovation models recently developed for computing systems may be helpful where the type of software is optional for the individual worker.

Automation↗
PubMed2002

Nucleotide sequence-based typing of bacteria and the impact of automation.

DNA-based typing methods are increasingly important for the characterisation of bacteria. They are used to monitor the epidemiology of pathogens with public health significance and also to help understand the evolution and population biology of bacteria. However, these methods require accuracy and reproducibility and are often of a high-throughput nature. Laboratory automation is therefore the key to the successful implementation of such methods. This review describes the impact of automation on DNA-based typing methods, particularly multi-locus sequence typing (MLST), and the method components that can be automated.

Automation↗
PubMed1995

Variability in absolute lymphocyte counts obtained by automated cell counters.

There is increasing interest in the absolute lymphocyte count. This is partly driven by the need to obtain absolute values for lymphocyte subsets such as absolute CD4+ counts in human immunodeficiency virus (HIV)-infected persons. The absolute total lymphocyte count is usually determined in the routine hematology laboratory on a separate sample from the same patient specimen and then combined with percentage results from flow cytometry to obtain the absolute value of the lymphocyte subsets. We have studied analytic variability in the absolute lymphocyte determination and compared it to the variability of the total white blood count (WBC). In a series of 524 specimens, four different automated methods were compared to each other and to the traditional eye count differential. The automated methods were four widely used automated cell counters (Technicon H*1, TOA NE8000, Coulter STKS, and Abbott CD3000). The results indicate that analytic variability in the absolute lymphocyte counts, due, primarily, to method variability, is significant and is larger than the variability typically observed on interlaboratory trials of relative CD4 counts. These method biases cannot easily be reduced by calibration, since the cell classification algorithms are built-in features of the various cell counters. Analytic variability of the absolute lymphocyte counts was found to be 12.4% compared with analytic variability of only 4.9% for total WBC counts on the same samples. Our data suggest that more precise results would be obtained if flow cytometry results expressed each phenotype as a fraction of the leukocytes as well as total lymphocytes. Conversion to absolute values could then be accomplished through determination of the total WBC in the routine hematology laboratory.

Automation↗
PubMed2004

Automated microscopy identifies estrogen receptor subdomains with large-scale chromatin structure unfolding activity.

BACKGROUND: Recently, several transcription factors were found to possess large-scale chromatin unfolding activity; these include the VP16 acidic activation domain, BRCA1, E2F1, p53, and the glucocorticoid and estrogen steroid receptors. In these studies, proteins were fluorescently labeled and targeted to a multimerized array of DNA sequences in mammalian cultured cells, and changes in the appearance and/or size of the array were observed. This type of experiment is impeded by the low throughput of traditional microscopy. METHODS: We report the application of automated microscopy to provide unattended, rapid, quantitative measurements of fluorescently labeled chromosome regions. RESULTS: The automated image collection routine produced results comparable to results previously obtained by manual methods and was significantly faster. Using this approach, we identified two subdomains within the E domain of estrogen receptor alpha capable of inducing large-scale chromatin decondensation. CONCLUSIONS: This work confirms that, like BRCA1, the activation function 2 region of the estrogen receptor has more than one distinct chromatin unfolding domain. In addition, we demonstrate the feasibility of using automated microscopy as a high-throughput screen for identifying modulators of large-scale chromatin folding. The Supplementary Material referred to in this article can be found at the CYTO Part A website (http://www.interscience.wiley.com/jpages/0196-4763/suppmat/v58A.html)

Animals↗
PubMed1992

Fluorescence chemistries for automated primer-directed DNA sequencing.

A major limitation in the applicability of automated DNA sequencing instruments has been the difficulty in using user-defined oligonucleotide primers which allow sequencing reactions to start at any specific point in a region of interest. Recently, new chemistries have become available for fluorescent labeling which will begin to facilitate the use of any oligonucleotide primer with automated DNA sequencers. In this report, we describe several methods for automated primer-directed DNA sequencing, and compare and discuss the relative merits and limitations of these methods.

Automation↗
PubMed2003

Comparison of automated in-gel digest methods for femtomole level samples.

A comparison of automated in-gel digestion methods for low picomolar to femtomolar levels of protein is presented. Gel spots with 4 pmol to 120 fmol of protein were stained with either Coomassie colloidal blue or SYPRO Ruby and digested using an automated platform. The sequence coverages and average peak intensities obtained from a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis are compared. Results show that methods using an acetonitrile extraction or digest times greater than the standard 4 h give no significant increase in peptide sequence coverage for automated digestion of low protein level samples. It is also shown that digests from SYPRO Ruby-stained gels show a greater improvement upon ZipTip cleanup than digests from Coomassie colloidal blue-stained gels. The digests from SYPRO Ruby-stained gels are also shown to give a higher average peptide intensity if a method with minimal gel plug washing is used.

Automation↗
PubMed2005

Comparison of manual and automated ELISA methods for serum ferritin analysis.

Serum ferritin concentration is a sensitive measure of body iron stores. The aim of this study was to compare the performance of two commercially available enzyme-linked immunoassays (ELISAs) for serum ferritin: a widely used manual assay kit (Spectro Ferritin MT), and a new fully automated assay (Immulite). We analyzed serum samples from Moroccan school-aged children (n=51) from a rural area with a high prevalence of iron deficiency anemia (IDA). Four replicates of each sample were analyzed using both assays. For the manual method, the interassay repeatability was 24%, 22%, and 11%, and intraassay precision was 18.3%, 9.2%, and 9.1% at increasing serum ferritin concentrations. Using the automated assay, the interassay repeatability was 7%, 6%, and 6%, and intraassay precision was 1.5%, 5.4%, and 5.5% at increasing serum ferritin concentrations. The two assays were well correlated (y=1.16x+1.83; r=0.98). However, the limits of agreement (LOAs) were wide, particularly at low concentrations. A comparison of the assay results with recommended cutoffs for serum ferritin generated sharply different estimates of the prevalence of iron deficiency (ID) in the sample. We conclude that the automated assay has several potential advantages compared to the manual method, including better precision, less operator dependence, and faster sample through-put.

Adolescent↗
PubMed2004

Automated detection and delineation of mitochondria in electron micrographs of human skeletal muscles.

Morphometric measurements of mitochondria in human skeletal muscles provide useful information relating to tissue oxidative energy production, nutrition, exercise, and aging. Morphometric data such as area, perimeter, long axis, and short axis can be obtained by delineating individual mitochondria in electron micrographs. However, manual counting and delineating of individual mitochondria is a formidable task. The purpose of this study was to develop a fully automated computer algorithm for quantifying mitochondrial morphometry in electron micrographs. The algorithm locates mitochondria with a two-dimensional matched filter and then traces the borders of individual mitochondria. The delineation is accomplished by edge detection along radial lines launched outwards from the center of each mitochondrion. Shape descriptors applied to delineated mitochondria are used to reject likely false-positive selections. The results show that the fully automated algorithm detects mitochondria with a false-positive rate of 2% and a false-negative rate of 36%. The errors are easily and rapidly corrected by user intervention using a second semiautomated delineation algorithm. Morphometric measurements collected with the automated algorithm are equivalent to those obtained manually by human experts. The algorithm significantly improves the speed of image analysis and it also provides copious quantities of high-quality mitochondrial morphometric data.

Algorithms↗