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Comparative analysis of 35S and lectin promoters in transgenic soybean tissue using an automated image acquisition system and image analysis.

Expression of the green fluorescent protein (gfp) gene, under regulatory control of either the constitutive 35S promoter or the developmentally-regulated lectin promoter was monitored and quantified using a newly-developed automated tracking system. The automated system consisted of a computer-controlled two-dimensional robotics table and a programmable image acquisition system, which was used to semi-continuously monitor gfp gene expression during development of transgenic soybean [Glycine max (L.) Merrill] somatic embryos. Quantitative analysis of GFP expression showed that, during somatic embryo proliferation and early development, expression of lectin-GFP was not detected. During late embryo development, expression of lectin-GFP gradually increased until the levels were similar to those of 35S-GFP. The use of an automated image collection system and image analysis facilitated the frequent monitoring and quantification of gfp gene expression on a large number of samples over an extended period of time.

Automation↗

Global left ventricular function in cardiac CT. Evaluation of an automated 3D region-growing segmentation algorithm.

The purpose was to evaluate a new semi-automated 3D region-growing segmentation algorithm for functional analysis of the left ventricle in multislice CT (MSCT) of the heart. Twenty patients underwent contrast-enhanced MSCT of the heart (collimation 16 x 0.75 mm; 120 kV; 550 mAseff). Multiphase image reconstructions with 1-mm axial slices and 8-mm short-axis slices were performed. Left ventricular volume measurements (end-diastolic volume, end-systolic volume, ejection fraction and stroke volume) from manually drawn endocardial contours in the short axis slices were compared to semi-automated region-growing segmentation of the left ventricle from the 1-mm axial slices. The post-processing-time for both methods was recorded. Applying the new region-growing algorithm in 13/20 patients (65%), proper segmentation of the left ventricle was feasible. In these patients, the signal-to-noise ratio was higher than in the remaining patients (3.2+/-1.0 vs. 2.6+/-0.6). Volume measurements of both segmentation algorithms showed an excellent correlation (all P<or=0.0001); the limits of agreement for the ejection fraction were 2.3+/-8.3 ml. In the patients with proper segmentation the mean post-processing time using the region-growing algorithm was diminished by 44.2%. On the basis of a good contrast-enhanced data set, a left ventricular volume analysis using the new semi-automated region-growing segmentation algorithm is technically feasible, accurate and more time-effective.

Aged↗

A system for automated procedure documentation.

Quantitative information regarding competency in performing procedures is invaluable in attesting to a house officer's abilities when assessments need to be made. Thus, the means by which procedures, proficiency, and experience can be recorded and organized in an automated fashion need not be any further away than the nearest micro-computer. The procedure documenting system (PDS) automates the documenting of procedures and quantitative information pertaining to these procedures, and shortens the time demanded of the house officer to document procedures from a mean of 5.5 to 1.9 min. This paper is a description of the work done to automate the recording of procedures performed by housestaff at a teaching hospital and the acceptance by housestaff. This computer program takes the specific information and places it into a specialized database so that house officers can have detailed documentation attesting to their proficiency in various procedures. In addition, the program director can access the database to document the quantity of procedures to which an individual house officer has attained competence.

Automation↗

The service sector revolution: the automation of services.

This paper describes the service sector revolution in the United States and covers the automation of 10 U.S. service industries. It discusses the implications of the simultaneous automation of goods and services and suggests action required to smooth the transition for the worker as the United States becomes an automated society.

Automation↗

N-acetyl[3H]leukotriene D4: a stable internal standard for automated BIO-FAST HPLC extraction and separation of LTE4 from human urine.

The BIO-FAST (Fully Automated Sample Treatment) HPLC can be used for the isolation and separation of leukotriene E4 (LTE4) from the urine of asthmatic patients. A chemically related leukotriene, N-acetyl[14,15-3H]leukotriene D4 (NAc[3H]LTD4), has been evaluated as an internal standard to allow full automation of the BIO-FAST method. NAcLTD4 is not a human metabolite, does not co-elute with endogenously produced LTs and is stable in native urine at 37 degrees C for at least 18 h. Recovery and stability studies were conducted by adding NAc[3H]LTD4 and [3H]LTE4 to the baseline urine of four asthmatic patients. Automated extraction of these four samples over 22 hours, using the BIO-FAST system, yielded recoveries of 80.5% (6.6 %CV, n = 12) and 72.4% (10.0 %CV, n = 12) for the NAc[3H]LTD4 and [3H]LTE4, respectively. The ratio of NAc[3H]LTD4 to [3H]LTE4 was 1.12 (6.3 %CV, n = 12) demonstrating the consistent relative extraction of these two leukotrienes.

Automation↗

Automation in manned spaceflight.

With the ever increasing complexity and use of advanced technologies in the development of equipment and experiments in spaceflight, the possibility offers itself to automating repetitious or very sensitive operations. The implementation of such automation lends itself well to teleoperations and the eventual introduction of artificial intelligence. In this paper the criteria for introducing automation are discussed, and its impacts examined. New problems such as command and control are discussed, with some guidelines suggested.

Artificial Intelligence↗

Automated metaphase finding: an assessment of the efficiency of the METAFER2 system in a routine mutagenicity assay.

The efficiency of the automated metaphase finding system METAFER2 is assessed in a routine mutagenicity assay using an aneuploid rat liver cell line treated with various promutagens. Data sets generated by automated and manual selection of metaphases are compared. It is demonstrated that METAFER2 routinely allows an efficient automatic identification of metaphases not only in lymphocyte preparations, but also in preparations from mammalian cell lines with varying chromosome numbers. Although larger slide areas are required for automated compared to manual metaphase scanning, the automatic system is faster by a factor of about 5. The interactive visual elimination of metaphases of insufficient quality is an easy and fast procedure. METAFER2 allows an unbiased selection of metaphases irrespective of their appearance as homogeneously stained first or harlequin-stained second division cells. Random selection of metaphases is neither influenced by various structural chromosome changes nor by increased frequencies of sister-chromatid exchanges.

Aneuploidy↗

Organizing and implementing a hospital-wide first-responder automated external defibrillation program: strengthening the in-hospital chain of survival.

First-responder automated external defibrillation (AED) in the hospital is consistent with the American Heart Association's (AHA) early defibrillation standard or care. With trained personnel and automated external defibrillators immediately available, early defibrillation should have a greater impact on survival than early cardiopulmonary resuscitation (CPR). Therefore, in our hospitals we modified basic life support to include automated external defibrillation (BLS-AED) for all personnel who are expected to respond to a cardiac arrest, with rapid defibrillation taking priority over CPR. We describe how we organized and implemented this hospital-wide first-responder BLS-AED program. Planning the process includes gaining support from key leaders who are responsible for resuscitation practice, and identifying the target audience of the training program. Hospital unit needs for AED or conventional defibrillation and equipment must be identified, the training program developed, and existing policies and procedures modified. Several barriers to implementation may exist. Education about the efficacy and safety of AED and experience once the BLS-AED program is in place can overcome attitudes and bias. Concerns about the cost of equipment and training must be addressed. Program evaluation may include patient issues such as measuring the time to the first defibrillation and patient outcome; as well as training and retention issues.

Adult↗

Automation and validation of the high-performance liquid chromatographic-radioimmunoassay method for the determination of lacidipine in plasma.

The automation and validation of the HPLC-radioimmunoassay (RIA) method for the determination of lacidipine are reported. The solid-phase extraction step was automated by the introduction of the ASPEC system. A two-column system was adopted for the HPLC purification. The RIA was converted from heterogeneous to homogeneous by the scintillation proximity assay system and automated using an automatic dilution system. All characteristics in terms of accuracy, precision, specificity, and linearity resulted similar to the manual version. The quantification limit was set to 40 pg/ml. The new version of the method increased the number of samples assayed per month two- to three-fold.

Antihypertensive Agents↗

Fully automated assay for the determination of GR117289X in human plasma using mixed mode solid-phase extraction and high-performance liquid chromatography with ultraviolet detection.

GR117289X, 3-(3-Bromo-2-[2-(1H-tetrazol-5-yl)-phenyl]-bezofuran-5-yl methyl)-2-butyl- 5-chloro-3H-imidazole-4-carboxylic acid, 1, is an angiotensin II receptor antagonist. A sensitive, accurate and precise assay for the determination of 1 in human plasma and validation data is described. The assay consists of a novel solid-phase extraction using Certify II followed by a reversed-phase gradient HPLC separation with UV detection. The extraction procedure has been fully automated by a Zymate XP robot and linked on-line to the HPLC system. The analytical range for the assay is 5-250 ng ml-1 over which the assay is linear and specific for 1 with respect to endogenous plasma components and its tetrazole N2 glucuronide, the major circulating metabolite 2. For the fully automated procedure the intra-assay data indicate a maximum bias and coefficient of variation across the calibration range of +8.0 and 9.4%, respectively. The inter-assay data indicate a maximum bias and coefficient of variation across the calibration range of +8.0 and 11.1%, respectively. The extraction efficiency of the assay was approximately 75%. Both a manual and fully automated assay were applied to the analysis of 1 in plasma of volunteers from a number of clinical studies. The assay has been shown to be robust in sustained use over several months.

Angiotensin Receptor Antagonists↗

Validation of automated behavioral test systems.

A positive control study was conducted as part of the ongoing validation program for developmental neurotoxicity testing in our laboratory using a standard battery of automated systems, consisting of rotorod, motor activity, acoustic startle, and two-way active avoidance. Female Sprague-Dawley rats were given 10 mg/kg diazepam (DZ) by SC injection or 20 mg/kg methimazole (MET) by gavage from gestation day 15 (DZ) or 17 (MET) through postpartum day 10; a group of control animals remained untreated. Offspring were assessed for growth, survival, developmental landmarks, and behavior. Although this study was considered useful for obtaining historical data, it offered few advantages in terms of validation of automated behavior test systems. Perinatal treatment with DZ resulted in no maternal toxicity and no adverse effects on growth or development of F1 offspring; a deficit in acoustic startle responding was the only behavioral effect observed. Treatment with MET resulted in maternal toxicity, reduced neonatal body weights, and developmental delays. Behavioral effects included impaired rotorod performance and acoustic startle responding (neonates), and enhanced motor activity and acoustic startle responding (young adults). However, effects on shuttle avoidance were not observed for either drug, and only one direction of behavioral effect occurred for the rotorod and motor activity systems. These results, as well as those from subsequent studies in our laboratory, suggest that it may be preferable to validate automated behavior systems using short-term studies in which young adult animals are treated directly with positive control agents.

Animals↗

An automated homogeneous method for quantifying polysorbate using fluorescence polarization.

An automated fluorescence polarization (FP) assay has been developed for the quantitation of polysorbate in bioprocess samples. Using the lipophilic probe 5-dodecanoylaminofluorescein (DAF), polysorbate concentrations above the critical micelle concentration can be quantified by the FP increase that results when DAF inserts into the detergent micelles. The specificity, accuracy, and precision of this assay were defined for samples obtained from vaccine purification processes. Spike recoveries were 98-106% for purified products and 110-120% for crude process intermediates. The coefficients of variation for intra- and interassay precision were less than 9 and 14%, respectively. Because of the operational simplicity of the assay, all of the assay steps from sample preparation to data reduction were automated on a Tecan liquid-handling workstation. The combination of a rapid assay and an automated format makes this method well suited to the routine analysis of samples from trial purification processes which are carried out during the development of a vaccine or therapeutic protein. This method should be adaptable for the quantitation of other detergents into which DAF will insert.

Automation↗

CT angiography of peripheral arterial bypass grafts: Accuracy and time-effectiveness of quantitative image analysis with an automated software tool.

RATIONALE AND OBJECTIVES: Qualitative analysis of computed tomography (CT) angiography data often is limited by intra- and interobserver variability. The purpose of this study was to evaluate the time-effectiveness and accuracy of a quantitative CT angiography data analysis using automated software in comparison with qualitative axial and coronal CT image reading in patients with peripheral bypass grafts. MATERIALS AND METHODS: Twenty-eight patients with 33 saphenous bypass grafts underwent 4-channel (n = 21) and 16-channel (n = 7) CT angiography. Two readers evaluated in consensus the CT data qualitatively on axial and coronal reconstructions and with the software regarding the presence of graft stenoses, aneurysmal changes, and arteriovenous fistulas. The time for data analysis was taken and the accuracy was compared with the results from digital subtraction angiography (DSA). RESULTS: No significant difference was present between data analysis time using axial and coronal CT images (4.9 +/- 1.5 minutes) and when using the software tool (5.5 +/- 1.4 minutes). Good (kappa = 0.652) to excellent (kappa = 1.000) intermodality agreement was present between qualitative and quantitative CT analysis regarding graft-related abnormalities. Sensitivity and specificity for diagnosing stenoses, aneurysms, and fistula did not differ significantly (P > .025) between qualitative CT image reading and the automated software tool. CONCLUSIONS: CT angiography analysis of peripheral bypass grafts using an automated software tool is similar regarding time-effectiveness and accuracy when compared with qualitative CT data analysis on axial and coronal images. It may assist in determining the significance of an abnormality and can yield objective morphometric data of vessel calibers.

Algorithms↗

Full automation of derivatization--solid-phase microextraction-gas chromatography-mass spectrometry with a dual-arm system for the determination of organometallic compounds in aqueous samples.

The determination of organometallic compounds in aqueous samples by in-vial derivatization and headspace solid-phase microextraction (SPME)-gas chromatography (GC)-mass spectrometry (MS) has been fully automated using a Twin PAL dual-arm robotic system. Linearity, accuracy, sensitivity for a series of n-methyl, n-ethyl, and n-phenyl metal substituted chloride compounds of tin, lead, and mercury were investigated. The automated method was compared to similar manual methods and improved precision, speed and throughput was achieved. By originally programming the Twin PAL dual-arm system with the supplier's software (Cycle Composer, Version 1.5.0) the arms on the robot were only able to work in sequence. However, in order to have a flexible system and exploit time efficiently the robotic arms must work simultaneously. This was accomplished by programming the robot with the new software package called Cruise Control 4-2 for Twin PALs. Compared to Cycle Composer, Cruise Control 4-2 enhanced the speed and throughput of the automated system further. In addition, with a built-in crash prevention protocol and an improved user interface a more user-friendly system was obtained.

Automation↗

Automated sample preparation based on the sequential injection principle. Solid-phase extraction on a molecularly imprinted polymer coupled on-line to high-performance liquid chromatography.

A molecularly imprinted polymer (MIP) prepared using caffeine, as a template, was validated as a selective sorbent for solid-phase extraction (SPE), within an automated on-line sample preparation method. The polymer produced was packed in a polypropylene cartridge, which was incorporated in a flow system prior to the HPLC analytical instrumentation. The principle of sequential injection was utilised for a rapid automated and efficient SPE procedure on the MIP. Samples, buffers, washing and elution solvents were introduced to the extraction cartridge via a peristaltic pump and a multi-position valve, both controlled by appropriate software developed in-house. The method was optimised in terms of flow rates, extraction time and volume. After extraction, the final eluent from the extraction cartridge was directed to the injection loop and was subsequently analysed on HPLC. The overall set-up facilitated unattended operation, operation and improved both mixing fluidics and method development flexibility. This system may be readily built in the laboratory and can be further used as an automated platform for on-line sample preparation.

Automation↗

Automated analysis of individual particles using a commercial capillary electrophoresis system.

Capillary electrophoretic analysis of individual submicrometer size particles has been previously done using custom-built instruments. Despite that these instruments provide an excellent signal-to-noise ratio for individual particle detection, they are not capable of performing automated analyses of particles. Here we report the use of a commercial Beckman P/ACE MDQ capillary electrophoresis (CE) instrument with on-column laser-induced fluorescence (LIF) detection for the automated analysis of individual particles. The CE instrument was modified with an external I/O board that allowed for faster data acquisition rates (e.g. 100 Hz) than those available with the standard instrument settings (e.g. 4 Hz). A series of eight hydrodynamic injections expected to contain 32 +/- 6 particles, each followed by an electrophoretic separation at -300 V cm(-1) with data acquired at 100 Hz, showed 28 +/- 5 peaks corresponding to 31.9 particles as predicted by the statistical overlap theory. In contrast, a similar series of hydrodynamic injections followed by data acquisition at 4 Hz revealed only 8 +/- 3 peaks suggesting that the modified system is needed for individual particle analysis. Comparison of electropherograms obtained at both data acquisition rates also indicate: (i) similar migration time ranges; (ii) lower variation in the fluorescence intensity of individual peaks for 100 Hz; and (iii) a better signal-to-noise ratio for 4 Hz raw data. S/N improved for 100 Hz when data were smoothed with a binomial filter but did not reach the S/N values previously reported for post-column LIF detection. The proof-of-principle of automated analysis of individual particles using a commercially available CE system described here opens exciting possibilities for those interested in the study and analyses of organelles, liposomes, and nanoparticles.

Automation↗

A novel automated method to measure total antioxidant response against potent free radical reactions.

OBJECTIVES: Oxidative damage of biomolecules occurs as a result of potent free radical reactions. In this study, a novel, colorimetric and fully automated method for measuring total antioxidant response (TAR) against potent free radical reactions is described. DESIGN AND METHODS: Potent free radical reactions were initiated with the production of hydroxyl radical (OH(*)) via Fenton reaction, and the rate of the reactions was monitored by following the absorbance of colored dianisidyl radicals. Ortho-dianisidine (10 mM) and ferrous ammonium sulfate (45 microM) were dissolved in KCl/HCl solution (75 mM, pH 1.8). This mixture was named as Reagent 1 and hydrogen peroxide solution (7.5 mM) as Reagent 2. The OH(*), produced by mixing of R1 and R2, oxidized o-dianisidine molecules into dianisidyl radicals, leading to a bright yellow-brown color development within seconds. Antioxidants, present in the sample, suppressed the color formation to a degree that is proportional to their concentrations. The method was applied to an automated analyzer and analytical performance characteristics of the assay were determined. RESULTS: Vitamin C and Trolox, reduced glutathione, bilirubin, uric acid and (+/-)-catechin solutions suppressed the color formation depending on their concentrations. Serum TAR against potent free radical reactions was lower in patients with chronic renal failure (1.13 +/- 0.21 mmol Trolox equiv./l) and was higher in the individuals with neonatal icterus (2.82 +/- 1.18 mmol Trolox equiv./l) than in healthy subjects (1.54 +/- 0.15 mmol Trolox equiv./l). CONCLUSIONS: The easy, inexpensive and fully automated method described can be used to measure TAR of samples against potent free radical reactions.

Antioxidants↗

A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation.

OBJECTIVES: To develop a novel colorimetric and automated direct measurement method for total antioxidant capacity (TAC). DESIGN AND METHODS: A new generation, more stable, colored 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS(*+)) was employed. The ABTS(*+) is decolorized by antioxidants according to their concentrations and antioxidant capacities. This change in color is measured as a change in absorbance at 660 nm. This process is applied to an automated analyzer and the assay is calibrated with Trolox. RESULTS: The novel assay is linear up to 6 mmol Trolox equivalent/l, its precision values are lower than 3%, and there is no interference from hemoglobin, bilirubin, EDTA, or citrate. The method developed is significantly correlated with the Randox- total antioxidant status (TAS) assay (r = 0.897, P < 0.0001; n = 91) and with the ferric reducing ability of plasma (FRAP) assay (r = 0.863, P < 0.0001; n = 110). Serum TAC level was lower in patients with major depression (1.69 +/- 0.11 mmol Trolox equivalent/l) than in healthy subjects (1.75 +/- 0.08 mmol Trolox equivalent/l, P = 0.041). CONCLUSIONS: This easy, stable, reliable, sensitive, inexpensive, and fully automated method described can be used to measure total antioxidant capacity.

Antioxidants↗