Application of the CYTOMIC 12 flow cytometric compact analyzer for automatic kinetic measurements

Flow cytometry, usually applied to cells which have time independent features, can also be used for kinetic experiments where the change of cell populations with time is investigated. Dedicated time sequencing programs written in Assembler and incorporated in the CYTOMIC 12 analyzer (4) are described. A sequence of 64 one parameter histograms can be automatically acquired and immediately displayed as a pseudo-two-parameter histogram. The acquisition time for each of the subsequent histograms can be selected between 1 and 32 seconds. Kinetics lasting up to 34 minutes are resolved into 64 time intervals. Two parameter kinetics can be resolved into 12 32 X 32 channel, two parameter histograms which are displayed and evaluated immediately on the analyzer screen in groups of 4 without using complicated list mode procedures. The standard CYTOMIC 12 software can be applied for processing and printing of the sequence distribution curves.     

Evaluation of cardiac assays on a benchtop chemiluminescent enzyme immunoassay analyzer, PATHFAST

Devices for cardiac immunoassay are widely available at point of care settings. The lateral flow method is the most popular solution for ease of use. But its major shortcomings, poor assay precision, and low analytical sensitivity may lead to false negative results. Therefore, confirmatory testing by a routine lab analyzer sometimes is necessary. In the current study, we evaluated the cardiac assays troponin I, MB isoenzyme of creatine kinase, myoglobin, and N-terminal pro brain natriuretic peptide on a chemiluminescent enzyme immunoassay analyzer, PATHFAST. All of the assays demonstrated correlation coefficients higher than 0.97 against the predicate devices along with good total precision (coefficients of variation <10%), and the troponin I assay showed analytical sensitivity in conformance with the guidelines of European Society of Cardiology/American College of Cardiology and National Academy of Clinical Biochemistry. We concluded that the system was rapid and easy to use without compromising analytical performance.     

Characterization of a prototype industrial on-line analyzer for bicarbonate/carbonate monitoring

In many biological reactors bicarbonate is the major species determining pH buffering capacity, or alkalinity. In anaerobic digesters bicarbonate levels should be within 10 to 50 mM for stable operation. Bicarbonate alkalinity in wastewater treatment processes in routinely measured off-line titrimetrically. Recently we have described the principle of a novel on-line method of measuring bicarbonate alkalinity. In the prototype device described here, a continuous stream (15 cm(3) min(-1)) of the substrate to be monitored was saturated with gaseous CO2, acidified by the addition of excess acid, and the rate of carbon dioxide evolution, proportional to the concentration of bicarbonate/carbonate in the liquid flow, continuously measured by a sensitive gas meter. The instrument was robust and its response was satisfactory for wastewater treatment process control applications, with linearity in the range 5 to 50 mM HCO3(-), a response time in the order of 30 min, and accuracy of the order of 7% in the concentration range 5 to 50 mM sodium bicarbonate. The device was not affected by interference from volatile fatty acids, does not make use of pH probes which in many wastes are subject to fouling, and may form the basis of a digester control strategy. (c) 1994 John Wiley & Sons, Inc.     

Evaluation of a noninvasive continuous cardiac output monitoring system based on thoracic bioreactance

Noninvasive cardiac output (CO) measurement can be useful in many clinical settings where invasive monitoring is not desired. Bioimpedance (intrabeat measurement of changes in transthoracic voltage amplitude in response to an injected high-frequency current) has been explored for this purpose but is limited in some clinical settings because of inherently low signal-to-noise ratio. Since changes in fluid content also induce changes in thoracic capacitive and inductive properties, we tested whether a noninvasive CO measurement could be obtained through measurement of the relative phase shift of an injected current (i.e., bioreactance). We constructed a prototype device that applies a 75-kHz current and determines the relative phase shift (dPhi/dt) of the recorded transthoracic voltage. CO was related to the product of peak dPhi/dt, heart rate, and ventricular ejection time. The preclinical study was done in nine open-chest pigs put on right heart bypass so that CO could be varied at known values. This was followed by a feasibility study in 27 postoperative patients who had a Swan-Ganz catheter (SGC). The measurements of noninvasive CO measurement and cardiopulmonary bypass pump correlated to each other (r = 0.84) despite the large variation in CO and temperatures. Similarly, in patients, mean CO values were 5.18 and 5.17 l/min as measured by SGC and the noninvasive CO measurement system, respectively, and were highly correlated over the range of values studied (r = 0.90). Preclinical and clinical data demonstrate the feasibility of using blood flow-related phase shifts of transthoracic electric signals to perform noninvasive continuous CO monitoring.     

Improved calculations of compactness and a reevaluation of continuous compact units

A new method for calculating compactness (Z) that uses look-up table-based algorithms for area and volume computations is introduced. These algorithms can be used in any iterative area orvolume calculation, are more than 1000 times faster than the originalalgorithms, and have equal or better precision. With the faster algorithms it is now possible to calculate the compactness of all continuous units in a protein, and to precisely locate the optimal compact units without the screening functions and limited resolution used previously. These methods have been incorporated into a fully automatic domain finding algorithm, and this method has been applied to the 21 proteins originally analyzed as well as 12 additional proteins. This method is robust, and yields similar units even when applied to coordinates of protein crystals grown under different experimental conditions. © 1993 Wiley-Liss, Inc.     

Evaluation of hprt mutant assay as a biomarker monitoring the specific environmental mutagen

To evaluate the availability of an hprtmutant assay for monitoring a specific environmental mutagen, the mutation effects of -irradiation and pentachlorophenol (PCP) on the hypoxanthine guanine phosphoriboxyl transferase (hprt) locus in a human T-cell culture system were analyzed in vitro. The assay of somatic mutation at the hprtlocus did not differentiate the characteristic effect of -irradiation from that produced by PCP, because both damaging agents induced the somatic mutations in a similar dose-dependent manner. Direct DNA sequencing showed that both damaging agents induced different mutation spectra in the hprtlocus of T-cells. The large deletions, which account for 75% of the analyzed mutants, were induced by -irradiation. By contrast, point mutations such as base substitutions rose up to 97% in the case of PCP-treated cells. It may be that 190 base pair and 444 base pair positions are hot spots induced by PCP. These results suggest that the hprtmutation spectrum can be used as a potential biomarker for assessing a specific environmental risk.     

Development of a turbidimetric immunoassay for on-line monitoring of proteins in cultivation processes

An on-line assay for a thermostable pullulanase and antithrombin III (AT III) is described. The assay is based on the formation of aggregates between the protein to be measured and antibodies raised against this protein. Assay automation was achieved by utilizing the flow injection analysis (FIA) principles. The apparatus, a stopped-flow, merging-zone manifold, is described in detail. Since the reaction used in an FIA system does not have to reach equilibrium, it was possible to reduce the time for an assay cycle to 2.5 min. A method for simulating cultivation conditions was developed for assay optimization. Using this method, a detection limit of I mg l⁻¹ together with a standard deviation of 1.5 was found. A sandwich ELISA was used as reference assay in the case of AT III and an enzymatic activity assay in the case of pullulanase. Correlation coefficients of 0.988 (AT III) and 0.976 (pullulanase) were determined. The turbidimetric assay was successfully used for pullulanase monitoring during a 240-h cultivation of Clostridium thermosulfurogenes.     

In vivo, continuous and automatic monitoring of extracellular ascorbic acid by microdialysis and on-line liquid chromatography

A system for in vivo, automatic, continuous monitoring of organ extracellular ascorbic acid in anesthetized rat is described. This system involves microdialysis perfusion and a LC system equipped with an electrochemical detector. Microdialysate, eluted from a microdialysis probe implanted in the brain cortex or in the left ventricular myocardium of anesthetized rats was collected in the sample loop of an on-line injector for direct injection onto the LC system. This automated method provides a shortened sample processing time. This system was utilized to investigate the effect of cerebral ischemia on cortex extracellular ascorbic acid and the effect of myocardial ischemia on left ventricular myocardium extracellular ascorbic acid in anesthetized rats. Basal ascorbic acid concentrations in the cortex and left ventricular myocardium ranged from 9.7 to 15.4 μM (mean±S.D. 12.7±2.5 μM from the results of eight rats) and from 9.3 to 36.0 μM (mean±S.D., 24.3±8.9 μM from the results of twelve rats), respectively. Cerebral ischemia significantly elevated ascorbic acid levels in the cortex extracellular space, while myocardial ischemia did not significantly alter ascorbic acid levels in the left ventricular myocardium extracellular space.     

Design of a new automatic chromatography system for efficient enzyme purification equipped with a time-shared multiple activity analyzer

A new system equipped with a computer-controlled multiple activity analyzer has been developed for the efficient purification of multiple enzymes. The system consists of the following units: conventional enzyme fractionation system with a peristaltic pump, liquid chromatographic column, fraction collector, and uv monitor; computer-operated uv-vis spectrophotometer equipped with a thermo-regulated metal block and a flow-through type silica cuvette; personal computer; dot matrix printer; cooling facility; and automatic sampling-mixing system. The whole system is operated by a newly designed time-sharing computer program for periodic and repetitive sampling of the column eluants containing multiple kinds of enzymes and of designated assay mixtures for each enzyme and for measurement of the initial velocity of spectrophotometric signals. For example, a mixture of aspartase (EC 4.3.1.1) and malate dehydrogenase (EC 1.1.1.39) and also a mixture of these two enzymes and glutamate dehydrogenase (EC 1.4.1.3 or EC 1.4.1.4) were analyzed by the above system using gel permeation chromatography, and the two or three enzyme activities were repeatedly monitored within 4 min. Based on the above results further possibilities for the application of the system for a variety of purposes are discussed.     

On-line monitoring of mammalian cell and yeast fermentations with a commercial biochemical analyzer

A commercial analyzer was tested for on-line monitoring of fermentations. A new sample block was constructed to effectively degas the fermentation broth. The robust analyzer accurately measured glucose up to 110 mmol/l and lactate up to 21 mmol/l at a frequency of 1 measurement per 2 minutes directly in suspensions of mammalian and yeast cells.     

Cellular and molecular mechanisms for rapid regression of atherosclerosis: from bench top to potentially achievable clinical goal

PURPOSE OF REVIEW: Decades of literature have unambiguously demonstrated regression and remodeling of atherosclerotic lesions, including advanced plaques. Recent insights into underlying mechanisms are reviewed. RECENT FINDINGS: Factors promoting regression include decreased apolipoprotein B-lipoprotein retention within the arterial wall, efflux of cholesterol and other harmful lipids from plaques, and emigration of lesional foam cells followed by entry of healthy phagocytes that remove necrotic debris and other plaque components. Cellular lipid efflux and foam cell emigration can occur surprisingly rapidly once the plaque milieu is improved. Lipid efflux and foam cell emigration each involve specific molecular mediators, many of which have been identified. Necrotic debris removal can be surprisingly comprehensive, with essentially full disappearance documented in animal models. SUMMARY: The essential prerequisite for regression is robust improvement in plaque milieu, meaning large plasma reductions in atherogenic apolipoprotein B-lipoproteins or brisk enhancements in 'reverse' lipid transport from plaque into liver. Importantly, the processes of regression are consistent with rapid correction of features characteristic of the rupture-prone, vulnerable plaques responsible for acute coronary syndromes. New interventions to lower apolipoprotein B-lipoprotein levels and enhance reverse lipid transport may allow regression to become a widespread clinical goal. Strategies based on recent mechanistic insights may facilitate further therapeutic progress.     

Evaluation of a malaria antibody enzyme immunoassay for use in blood screening

Transfusion-transmitted malaria is rare, but it may produce severe problem in the safety of blood transfusion due to the lack of reliable procedure to evaluate donors potentially exposed to malaria. Here, we evaluated a new enzyme-linked immunosorbent assay malaria antibody test (ELISA malaria antibody test, DiaMed, Switzerland) to detect antibodies to Plasmodium vivax (the indigenous malaria) in the blood samples in the Republic of Korea (ROK). Blood samples of four groups were obtained and analyzed; 100 samples from P.vivax infected patients, 35 from recovery patients, 366 from normal healthy individuals, and 325 from domestic travelers of non-endemic areas residents to risky areas of ROK. P.vivax antibody levels by ELISA were then compared to the results from microscopic examination and polymerase chain reaction (PCR) test. As a result, the ELISA malaria antibody test had a clinical sensitivity of 53.0% and a clinical specificity of 94.0% for P.vivax. Twenty out of 325 domestic travelers (6.2%) were reactive and 28 cases (8.6%) were doubtful. Of the reactive and doubtful cases, only two were confirmed as acute malaria by both microscopy and PCR test. Thus we found that the ELISA malaria antibody test was insufficiently sensitive for blood screening of P.vivax in ROK.     

Real-time monitoring and automatic density control of large-scale microalgal cultures using near infrared (NIR) optical density sensors

Signals from near infrared (NIR) light transmittance sensors were used for both real-time monitoring of algal biomass density in growing mass cultures (200l tubular biofences), and also as feedback in a system that controlled the density of the culture by automatic injection of fresh growth medium. When operated in a semi-continuous production mode between predefined density values, diurnal growth patterns were recorded on-line that provided information on the dynamics of the microalgal cultures with respect to environmental conditions. The bioreactor system was also programmed to operate in constant biomass density mode, thereby maintaining the culture at the optimal population density (OPD), and sustaining high biomass production levels. The system has potential for operating a dynamic density set point for microalgal cultures where the optimal population density varies as a function of ambient growing conditions.     

Construction of a panel of glucose indicator proteins for continuous glucose monitoring

The development of implantable glucose sensors for use in diabetes treatment has been pursued for decades. However, enzyme-based glucose sensors often fail in vivo. In our previous work, we engineered a novel glucose indicator protein (GIP) that can sense glucose without relying on any enzymes and cofactors. Nevertheless, this GIP is unsuitable for blood glucose monitoring due to its low dissociation constant. Here, we report a novel approach to creating a new GIP that can be used to monitor blood glucose level. By disrupting pi-pi stacking around GIP's glucose binding site through site-directed mutagenesis, we showed that GIP's dissociation constant can be manipulated from 0.026 mM to 7.86 mM. This approach yielded four GIP mutants. We showed that one of the mutants can be used to detect glucose from 0 to 32 mM, while another mutant can be employed to visualize intracellular glucose (0-200 μM) within living cells through FRET imaging microscopy measurement.     

Evaluation of a competitive antibody enzyme immunoassay for specific diagnosis of Chagas' disease

A competitive enzyme immunoassay based on the use of a monoclonal antibody (MAb) specific for "component 5" of Trypanosoma cruzi was evaluated. The antigenicity and immunogenicity of this component has been observed in natural and experimental infections. The studies were conducted in an area of Bolivia where mixed infections with Leishmania braziliensis are frequent and present a problem in the accurate diagnosis of T. cruzi infections. The specificity and sensitivity of this assay as compared to the indirect immunofluorescence and ELISA tests were demonstrated. The present test has proved to be more specific than the immunofluorescence and ELISA tests.     

Activation analysis of human hair as a tool for environmental pollution monitoring

Recent development and uses of neutron activation techniques for human hair analyses are reviewed. The method of neutron activation analysis (NAA) appears to have the potential to be used as a tool for environmental pollution monitoring. Principally, two types of NAA procedure are in use nowadays for multielement analyses of human scalp hair. The more common of these is the method of instrumental neutron activation analysis (INAA), consisting of a single short-term (3-10 hours) exposure of hair to a beam of neutrons in a nuclear reactor, followed by two measurements of gamma-ray spectra at 2-3 days and 3-4 weeks after the end of irradiation. The following microelements can be commonly determined by this type of activation procedure: As, Au, Br, Cu, K, La, Na, Sb, Sm, Co, Cr, Cs, Fe, Hg, Rb, Sc, Se and Zn. The other of the two procedures involves the use of radiochemical separation techniques and is employed for quantitative determinations of elements that are not easily determined by INAA (Mo, Cd, Ni, etc.), or in cases where there is a need to achieve the lowest possible limits of analytical determination. The accuracy of NAA techniques is strongly dependent on the hair sampling and hair sample processing methods used. The analytical error of this method may vary within the range of 5-15%. Its applicability as a tool for monitoring the environmental pollution level is here demonstrated on an example of groups of individuals living in the areas differing by the degree of environmental pollution. The use of other biopsy materials, such as e.g. mammalian hair, for the purpose of environmental exposure monitoring is also considered in this review.     

Reagentless and regenerable immunosensor for monitoring of immunoglobulin G based on non-separation immunoassay

Based on the enhancement of fluorescein isothiocyanate (FITC) fluorescence caused by reactions between proteins, we developed a reagentless, regenerable and rapid immunosensing system to determine immunoglobulin G (IgG). Fluorescence intensity of the immobilized FITC depends on IgG concentration, ranging from 10 to 50 microg/ml, specifically, even with co-existing proteins. The response time is 30 min during steady-state measurement and is less than a minute during transient measurement. When the FITC-labeled protein A binds to IgG, the surrounding atmosphere of FITC becomes hydrophobic. Since the fluorescence intensity of fluorescent substances generally increases at a hydrophobic environment, FITC fluorescence intensity increases with the concentration of protein A bonding to IgG. This system is regenerable because the fluorescence enhancement repeatedly occurs every time the immobilized fluorescent reagent is immersed in sample solutions.