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PubMed1979

New developments in analysis of isoenzymes separated by "high-performance" liquid chromatography.

We have developed two enzyme analyzers for use in "high-performance" liquid chromatography. In both systems two detectors are used, placed after the column effluent has been combined with assay reagent. In one system, an absorbance detector is placed before and after a post-column reaction coil. Peaks observed at one detector are subtracted from those at the other, to produce a two-point measurement of enzyme activity. The linear dynamic range was 17--1700 U/L for lactate dehydrogenase (EC 1.1.1.27). In the other system, two reaction coils were used and a single fluorescence detector was placed at the end of each coil. These coils were kept at different temperatures, and an automated switching valve diverted equal amounts of column effluent and reagent into both coils. The fluorescence readings were then subtracted to produce a differential measurement of enzyme activity. The linear dynamic range was 20--1000 U/L. We used both systems to chromatographically analyze lactate dehydrogenase isoenzymes, and could separately determine both the distribution and activity of sample isoenzymes.

Autoanalysis↗
PubMedDate not supplied

Contrast encoding in retinal bipolar cells: current vs. voltage.

To investigate the influence of voltage-sensitive conductances in shaping light-evoked responses of retinal bipolar cells, whole-cell recordings were made in the slice preparation of the tiger salamander, Ambystoma tigrinum. To study contrast encoding, the retina was stimulated with 0.5-s steps of negative and positive contrasts of variable magnitude. In the main, responses recorded under voltage- and current-clamp modes were remarkably similar. In general agreement with past results in the intact retina, the contrast/response curves were relatively steep for small contrasts, thus showing high contrast gain; the dynamic range was narrow, and responses tended to saturate at relatively small contrasts. For ON and OFF cells, linear regression analysis showed that the current response accounted for 83-93% of the variance of the voltage response. Analysis of specific parameters of the contrast/response curve showed that contrast gain was marginally higher for voltage than current in three of four cases, while no significant differences were found for half-maximal contrast (C50), dynamic range, or contrast dominance. In sum, the overall similarity between current and voltage responses indicates that voltage-sensitive conductances do not play a major role in determining the shape of the bipolar cell's contrast response in the light-adapted retina. The salient characteristics of the contrast response of bipolars apparently arise between the level of the cone voltage response and the postsynaptic current of bipolar cells, via the transformation between cone voltage and transmitter release and/or via the interaction between the neurotransmitter glutamate and its postsynaptic receptors on bipolar cells.

Animals↗
PubMed1996

Quantification of HIV-1 RNA in plasma: comparable results with the NASBA HIV-1 RNA QT and the AMPLICOR HIV monitor test.

We investigated and compared the reproducibility, accuracy, detection limits, and dynamic ranges of two commercial kits for quantification of RNA viral load using a titrated virus stock (laboratory strain HIV-1 IIIB) and 107 plasma samples of 25 HIV-1-infected patients. The high reproducibility of both methods (SD = 0.2-0.3 log for both methods) allowed reliable detection of a 0.5 log change in RNA viral load. Both methods had a similar detection limit (at least 10(3) RNA copies/ml plasma) and a dynamic range that extended over a 5 log (AMPLICOR) or a 6 log (NASBA) range of HIV-1 input. For HIV-1 IIIB, the viral load was compatible with measurements of virus-associated p24 antigen. For 21 patients (91 samples), the RNA viral load was similar with both methods differing by no more than 0.5 log. For four patients, the difference in viral load between the two methods was > 0.5 log for all 16 samples. For three of these patients, this could be explained by mismatches with primers or probes in the gag sequence: there was no correlation to the viral subtype. The RNA viral load determination was highly sensitive compared with p24 antigen measurement (> 95% of patients had a detectable viral load vs. 40% who had a detectable p24 level), but in the p24-positive samples the correlation between the antigen level and the RNA viral load was of only borderline significance. We also found that the viral RNA in whole blood was stable for at least 48 h during transport at room temperature. These observations show that both the NASBA HIV-1 RNA QT test and the AMPLICOR HIV monitor test are reliable parameters of the viral load, with great promise for their use as potential surrogate markers.

Base Sequence↗
PubMed2004

Fully automated, internally controlled quantification of hepatitis B Virus DNA by real-time PCR by use of the MagNA Pure LC and LightCycler instruments.

We report on the development of a fully automated real-time PCR assay for the quantitative detection of hepatitis B virus (HBV) DNA in plasma with EDTA (EDTA plasma). The MagNA Pure LC instrument was used for automated DNA purification and automated preparation of PCR mixtures. Real-time PCR was performed on the LightCycler instrument. An internal amplification control was devised as a PCR competitor and was introduced into the assay at the stage of DNA purification to permit monitoring for sample adequacy. The detection limit of the assay was found to be 200 HBV DNA copies/ml, with a linear dynamic range of 8 orders of magnitude. When samples from the European Union Quality Control Concerted Action HBV Proficiency Panel 1999 were examined, the results were found to be in acceptable agreement with the HBV DNA concentrations of the panel members. In a clinical laboratory evaluation of 123 EDTA plasma samples, a significant correlation was found with the results obtained by the Roche HBV Monitor test on the Cobas Amplicor analyzer within the dynamic range of that system. In conclusion, the newly developed assay has a markedly reduced hands-on time, permits monitoring for sample adequacy, and is suitable for the quantitative detection of HBV DNA in plasma in a routine clinical laboratory.

Automation↗
PubMed2001

Comparison between liquid chromatography-time of-flight mass spectrometry and selected reaction monitoring liquid chromatography-mass spectrometry for quantitative determination of idoxifene in human plasma.

This study compares HPLC electrospray time-of-flight mass spectrometry and selected reaction monitoring (SRM) LC-MS for high throughput quantitative determination of a small molecule drug in biological samples. A high throughput LC-MS method was developed for quantitatative determination of idoxifene in human plasma and the evaluation was accomplished with the cross-validation of the developed LC-MS method between the time-of-flight mass spectrometer, and a triple quadrupole mass spectrometer operated in the SRM mode. A simple one-step semi-automated 96-well liquid-liquid extraction procedure was used to prepare 96 samples in approximately 30 min and a rapid gradient was used to shorten the LC run time. Time-of-flight mass spectrometry provides acquisition of full-scan mass spectra and extracted ion current chromatograms, which may be extracted from the total ion current chromatogram for peak area determination. The limit of quantitation for idoxifene in human plasma obtained with the time-of-flight mass spectrometer was 5 ng/ml based on 100-microl aliquots of human plasma, and the linear dynamic range was from 5 ng/ml to 2000 ng/ml. The quantitative LC-MS results from the time-of-flight mass spectrometer demonstrated that precision did not exceed 7.1% and accuracy did not exceed 1.7% with reference to quality control samples at three concentration levels in replicates of six. In contrast, the limit of quantitation for idoxifene in human plasma using a tandem triple quadrupole mass spectrometer was 0.5 ng/ml with a linear dynamic range to 1000 ng/ml. The results from the triple quadrupole instrument show that the precision did not exceed 2.2% and accuracy did not exceed 2.9%. The overall results suggest time-of-flight mass spectrometry may be a viable technique for high throughput bioanalytical work for the quantitative determination of a representative small molecule drug in the low ng/ml range in human plasma.

Calibration↗
PubMed1994

Development of Non-Intensified Charge-Coupled Device Area X-ray Detectors.

Area X-ray detectors based on charge-coupled device imagers can provide excellent performance in terms of spatial resolution, sensitivity and dynamic range. Improvements in the fabrication of the primary converter, either scintillator or phosphor, mean that it is becoming possible to dispense with prestorage intensifiers and still provide outstanding low-level signal performance. Structured CsI scintillators are presented as one method of providing high efficiency and excellent spatial resolving power for this primary converter. Characterization of detector performance, in terms of such parameters as the detective quantum efficiency, point-spread function and dynamic range, needs to be directly related to the specific application of the detector. This contribution emphasizes the interplay of these parameters in the optimum design of detector systems. Performance prediction, based on measurements taken from prototype development systems, illustrates how such detectors will meet the exacting requirements of macromolecular crystallography.

Journal Article↗
PubMed1993

Dynamic changes in the receptive field properties of spinal cord neurons with ankle input in rats with chronic unilateral inflammation in the ankle region.

The aim of this study was to determine the discharge and receptive field properties of spinal cord neurons with ankle input in spinal segments L4-6 in the rat, both under control conditions and during the course of an adjuvant-induced unilateral inflammation in the ankle. The extent of receptive fields in the skin and deep tissue was assessed using brush, pinch and compression stimuli. Neurons were categorized as nociceptive-specific or wide-dynamic-range neurons on the basis of their response thresholds and responses to suprathreshold stimuli. At all stages of inflammation (2, 6, 13 and 20 days post inoculation) the population of neurons with ankle input showed differences from the population of neurons with ankle input in control rats. There was a reduction in the number of neurons that appeared as nociceptive specific and a concomitant increase in the number of neurons showing a wide-dynamic-range response profile. The receptive fields of the neurons with ankle input were markedly larger in rats with inflammation in the ankle region and mainly spread proximally on the ipsilateral hindlimb and also to the abdomen and tail in some cases. There was also an increase in the number of neurons with contralateral excitatory inputs. The mechanical thresholds at the ankle joint and proximal parts of the ipsilateral hindlimb were less in arthritic rats than in controls. The proportion of spontaneously active neurons was also increased in rats during the initial and later stages of inflammation, although there was no significant increase in the mean spontaneous discharge frequency. These data show that there are long-term changes in the receptive field and response properties of neurons in intact rats with chronic unilateral adjuvant-induced inflammation similar to those described previously in spinal cats with acute inflammation (Neugebauer and Schaible 1990). It is presumed that similar afferent and spinal mechanisms are at work under acute and chronic inflammatory conditions which produce hyperexcitability in spinal neurons with joint input.

Animals↗
PubMed2005

Improvement of a FRET-based indicator for cAMP by linker design and stabilization of donor-acceptor interaction.

Förster resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for a variety of intracellular molecular events. Often, however, the poor dynamic range of such reporters prevents detection of subtle but physiologically relevant signals. Here we present a strategy for improving FRET efficiency between donor and acceptor fluorophores in a green fluorescent protein (GFP)-based protein indicator for cAMP. Such indicator is based on protein kinase A (PKA) and was generated by fusion of CFP and YFP to the regulatory and catalytic subunits of PKA, respectively. Our approach to improve FRET efficiency was to perform molecular dynamic simulations and modelling studies of the linker peptide (L11) joining the CFP moiety and the regulatory subunit in order to define its structure and use this information to design an improved linker. We found that L11 contains the X-Y-P-Y-D motif, which adopts a turn-like conformation that is stiffly conserved along the simulation time. Based on this finding, we designed a new linker, L22 in which the YPY motif was doubled in order to generate a stiffer peptide and reduce the mobility of the chromophore within the protein complex, thus favouring CFP/YFP dipole-dipole interaction and improving FRET efficiency. Molecular dynamic simulations of L22 showed, unexpectedly, that the conformational behaviour of L22 was very loose. Based on the analysis of the three principal conformational states visited by L22 during the simulation time, we modified its sequence in order to increase its rigidity. The resulting linker L20 displayed lower flexibility and higher helical content than L22. When inserted in the cAMP indicator, L20 yielded a probe showing almost doubled FRET efficiency and a substantially improved dynamic range.

Amino Acid Sequence↗
PubMed2005

Use of a mixed tissue RNA design for performance assessments on multiple microarray formats.

The comparability and reliability of data generated using microarray technology would be enhanced by use of a common set of standards that allow accuracy, reproducibility and dynamic range assessments on multiple formats. We designed and tested a complex biological reagent for performance measurements on three commercial oligonucleotide array formats that differ in probe design and signal measurement methodology. The reagent is a set of two mixtures with different proportions of RNA for each of four rat tissues (brain, liver, kidney and testes). The design provides four known ratio measurements of >200 reference probes, which were chosen for their tissue-selectivity, dynamic range coverage and alignment to the same exemplar transcript sequence across all three platforms. The data generated from testing three biological replicates of the reagent at eight laboratories on three array formats provides a benchmark set for both laboratory and data processing performance assessments. Close agreement with target ratios adjusted for sample complexity was achieved on all platforms and low variance was observed among platforms, replicates and sites. The mixed tissue design produces a reagent with known gene expression changes within a complex sample and can serve as a paradigm for performance standards for microarrays that target other species.

Animals↗
PubMed2003

Principles of image processing in digital chest radiography.

Image processing has a major impact on image quality and diagnostic performance of digital chest radiographs. Goals of processing are to reduce the dynamic range of the image data to capture the full range of attenuation differences between lungs and mediastinum, to improve the modulation transfer function to optimize spatial resolution, to enhance structural contrast, and to suppress image noise. Image processing comprises look-up table operations and spatial filtering. Look-up table operations allow for automated signal normalization and arbitrary choice of image gradation. The most simple and still widely applied spatial filtering algorithms are based on unsharp masking. Various modifications were introduced for dynamic range reduction and MTF restoration. More elaborate and more effective are multi-scale frequency processing algorithms. They are based on the subdivision of an image in multiple frequency bands according to its structural composition. This allows for a wide range of image manipulations including a size-independent enhancement of low-contrast structures. Principles of the various algorithms will be explained and their impact on image appearance will be illustrated by clinical examples. Optimum and sub-optimum parameter settings are discussed and pitfalls will be explained.

Humans↗
PubMed1982

A new chromogenic amylase method compared with two established methods.

We tested a new amylase reagent involving chromogenic substrates (Pantrak, Calbiochem-Behring) for precision, dynamic range, and susceptibility to potential interferences. Two p-nitrophenyl-alpha-maltaosides are used as substrates, which amylase cleaves to shorter-chain p-nitrophenyl maltaosides, the latter then yielding p-nitrophenol from the activity of alpha-glucosidase. A series of 100 specimens were tested by Pantrak and two established methodologies. In both cases, correlation was excellent. Precision was good for all methods; CVs for Pantrak were 3.0 to 4.5%. The dynamic range of Pantrak extended to 800 U/L. Above-normal quantities of triglyceride, hemoglobin, or bilirubin did not cause spurious results, but anticoagulants that bind divalent cations should not be used. The Pantrak method has desirable analytical features and is easy to use.

Amylases↗
PubMedDate not supplied

Light photon equalization and its application to X-ray film digitization.

The principle of light photon equalization (LPE) is to modulate the light source during X-ray film digitization so that the dynamic range of the transmitted light into a photo-detector can be precisely controlled to obtain improved signal-to-noise ratio (SNR). The modulation of the light source is derived from a smoothed image of the original digitized image. The final output digital image is reconstructed by convoluting the modulation function with the resultant LPE image. The LPE was implemented in a laser film digitizer as a prototype. The results shows that, although the dynamic range of X-ray films is about 10(4):1, the range of the transmission light into a photo detector was reduced to 10(2):1 with this technique. Also the SNR improved by a maximum factor of five in the lung area of a digitized chest radiograph.

Algorithms↗
PubMed2002

Development and assessment of a novel real-time PCR assay for quantitation of HBV DNA.

HBV DNA quantitation is used extensively for the monitoring of treatment of hepatitis B virus (HBV) infection. The aim of this study was to develop a highly sensitive and reproducible real-time PCR (RTD-PCR) assay for the quantitation of HBV DNA using the LightCycler system. The performance of this assay was assessed by analyzing serial dilutions of HBV genomic DNA of known concentration and the lower limit of detection was found to be 1 DNA copy/reaction. By using serial dilutions of plasmid standard, RTD-PCR was determined to quantify HBV DNA in a 10-log10 dynamic range. RTD-PCR was found to be more sensitive than the commercially available tests such as the Quantiplex HBV DNA and the AMPLICOR HBV MONITOR assays. The median coefficient of variation of interexperimental variability was 3.2%. The HBV DNA values obtained with RTD-PCR were highly correlated with assays available commercially. These findings suggest that our RTD-PCR assay combines high sensitivity and reproducibility for HBV DNA quantitation in an incomparable high dynamic range of quantitation.

DNA, Viral↗
PubMed1987

Psychophysical studies relevant to the design of a digital electrotactile speech processor.

Psychophysical tests were carried out to investigate the perception of electrocutaneous stimuli delivered to the digital nerve bundles. The tests provided data for defining the operating range of a tactile aid for patients with profound-to-total hearing loss, as well as the individual differences between subjects and the information that could be transmitted. Monopolar biphasic constant current pulses with variable pulse widths were used. Threshold pulse widths varied widely between subjects and between fingers for the same subject. Thresholds were reasonably stable, but maximum comfortable levels increased with time. Perceived intensity was weakly dependent on pulse rate. Absolute identification of stimuli differing in pulse width gave information transmissions from 1.3-2.1 bits, limited by the dynamic ranges of the stimuli (3-17 dB). Stimuli from electrodes placed on either side of each finger were identified easily by all subjects. Absolute identification of stimuli differing in pulse rate gave information transmissions from 0.5-2.0 bits. Difference limens for pulse rate varied between subjects and were generally poor above 100 pps. On the basis of the results, an electrotactile speech processor is proposed, which codes the speech amplitude as pulse width, the fundamental frequency as pulse rate, and the second formant frequency as electrode position. Variable performances on tasks relying on amplitude and fundamental frequency cues are expected to arise from the intersubject differences in dynamic range and pulse rate discrimination. The psychophysical results for electrotactile stimulation are compared with previously published results for electroauditory stimulation with a multiple-channel cochlear implant.

Communication Devices for People with Disabilities↗
PubMed1999

Xenon has greater inhibitory effects on spinal dorsal horn neurons than nitrous oxide in spinal cord transected cats.

UNLABELLED: Xenon (Xe) suppresses wide dynamic range neurons in cat spinal cord to a similar extent as nitrous oxide (N2O). The antinociceptive action of N2O involves the descending inhibitory system. To clarify whether the descending inhibitory system is also involved in the antinociceptive action of Xe, we compared the effects of Xe on the spinal cord dorsal horn neurons with those of N2O in spinal cord-transected cats anesthetized with alpha-chloralose and urethane. We investigated the change of wide dynamic range neuron responses to touch and pinch by both anesthetics. Seventy percent Xe significantly suppressed both touch- and pinch-evoked responses in all 12 neurons. In contrast, 70% N2O did not show significant suppression in touch- and pinch-evoked responses. These results suggest that the antinociceptive action of Xe might not be mediated by the descending inhibitory system, but instead may be produced by the direct effect on spinal dorsal horn neurons. IMPLICATIONS: Xenon (Xe) is an inert gas with anesthetic properties. We examined the antinociceptive effects of Xe and nitrous oxide (N2O) in spinal cord-transected cats. Our studies indicate that Xe has a direct antinociceptive action on the spinal cord that is greater than that of N2O.

Anesthetics, Inhalation↗
PubMed2001

Flow-through UV spectrophotometric sensor for determination of (acetyl)salicylic acid in pharmaceutical preparations.

The solid phase spectrophotometry technique, in which the absorbance of the species of interest sorbed on a solid support is measured directly, was applied to the determination of salicylic acid using flow injection-analysis. Salicylic acid was determined by monitoring of its intrinsic absorbance at 297 nm sorbed on Sephadex QAE A-25 resin placed in an appropriate flow-through cell. The method proposed improves the selectivity compared with the corresponding solution-phase method and the sensitivity is increased by a factor of 30 or more. The flow-through sensor proposed allows working with several calibration lines simply by varying the sample volume injected. Thus, linear dynamic ranges from 1 to 20 and from 2 to 40 microg ml(-1) can be obtained by using 1000 and 300 microl, respectively, with detection limits being 0.064 and 0.135 microg ml(-1). Relative Standard Deviations (RSDs) of 0.52 and 0.38%, and sampling frequencies of 18 and 25 h(-1), respectively, were also achieved. The sensor also allows the indirect determination of acetylsalicylic acid previous hydrolysis on-line to salicylic acid. For acetylsalicylic acid, a linear dynamic range from 5 to 120 microg ml(-1) and 25 h(-1) of sampling frequency (300 microl of sample volume) were obtained. The proposed flow-through sensor has been successfully applied to the determination of both analytes in pharmaceutical preparations.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2000

Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC.

Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca2+ (Kd approximately 20 microM, 6 microM and 12 microM respectively) and selectivity over Mg2+ (Kd approximately 2 mM for mag-Fura-2, > 10 mM for Fura-2FF and BTC). Among the tested indicators, BTC was limited by a modest dynamic range upon Ca2+ binding, susceptibility to photodamage, and sensitivity to alterations in pH. All three indicators bound other metal ions including Zn2+, Cd2+ and Gd3+. Interestingly, only in the case of BTC were spectral differences apparent between Ca2+ and other metal ions. For example, the presence of Zn2+ increased BTC fluorescence 6-fold at the Ca2+ isosbestic point, suggesting that this dye may be used as a fluorescent Zn2+ indicator. Fura-2FF has high specificity, wide dynamic range, and low pH sensitivity, and is an optimal low-affinity Ca2+ indicator for most imaging applications. BTC may be useful if experimental conditions require visible wavelength excitation or sensitivity to other metal ions including Zn2+.

Animals↗
PubMed2005

Comprehensive comparison of the VERSANT HIV-1 RNA 3.0 (bDNA) and COBAS AMPLICOR HIV-1 MONITOR 1.5 assays on 1,000 clinical specimens.

BACKGROUND: Plasma human immunodeficiency virus type 1 (HIV-1) RNA level is an important parameter for patient management, yet viral load assays from different manufacturers are not standardized. OBJECTIVES AND STUDY DESIGN: In this study, we evaluated the concordance between test results obtained for 1,000 plasma specimens collected from HIV-1-infected individuals measured with the VERSANT HIV-1 RNA 3.0 assay (bDNA) and the COBAS AMPLICOR HIV-1 MONITOR 1.5 test (PCR). We compared viral load values obtained by each of these assays throughout their dynamic ranges, with particular focus on samples with low viral load (i.e. 50-250 copies/mL), and calculated the estimated distribution of distinct plasma viral load levels for the entire study population modeled from the data observed in the study. RESULTS: We found that these two assays show excellent agreement, with a correlation (R(2)) of 0.957 and a slope of 1.004. The mean difference in viral load values between the two assays was less than 0.10-log(10) throughout the dynamic range and 98.2% of all samples had bDNA and PCR results within 0.5-log(10) of each other, a difference that is within the range considered to be a minimal change in plasma viremia. Moreover, the two assays show very similar results across all assay ranges tested. The estimated prevalence of samples with results <50 copies/mL, 50-250 copies/mL, and 250-500,000 copies/mL were 41.6%, 7.7%, and 49.7%, respectively, by the bDNA assay, and 42.4%, 6.9%, and 50.7%, respectively, by the PCR assay. CONCLUSION: Based on our findings from 1,000 clinical specimens, we do not see the need to re-establish a baseline value or apply a conversion factor when switching from one assay to the other. Since the majority of our patient population likely is infected with subtype B virus, it is unclear if our findings will apply to other patient populations with a greater incidence of infection with non-B subtypes.

Branched DNA Signal Amplification Assay↗