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PubMed1999

High-throughput real-time reverse transcription-PCR quantitation of hepatitis C virus RNA.

We describe a rapid and reproducible method for assessment of the hepatitis C virus (HCV) load in serum samples. The method combines Taqman technology (Roche) and the ABI Prism 7700 (Perkin Elmer) real-time sequence detection system. We have optimized a single-tube reverse transcription-PCR (RT-PCR) that contains a dual-labeled fluorogenic probe to quantify the 5' noncoding region (5' NCR) of HCV. The probe contains a fluorescent reporter at the 5' end and a fluorescent quencher at the 3' end. The use of such a probe combined with the 5'-3' nuclease activity of Taq polymerase allows direct quantitation of the PCR product by the detection of a fluorescent reporter released in the course of the exponential phase of the PCR. For accurate quantitation of the number of copies of HCV in samples containing unknown quantities, we have used serial dilutions of a synthetic 5' NCR RNA standard of HCV that was previously quantified with an isotopic tracer. The method has a 5-log dynamic range (10(3) to 10(7)). The coefficient of regression of the standard curve was, on average, 0.98. The intra-assay and the interassay coefficients of variation of the threshold cycle were 1% and 6.2%, respectively. Seventy-nine RNA samples from the sera of infected patients were quantified by this method. Comparison of the results with those obtained by other quantitation methods (the Quantiplex 2.0 branched-DNA assay and the Superquant assay from the National Genetics Institute) revealed a significant correlation with all of the results. The mean values were also statistically comparable. In conclusion, the high sensitivity, simplicity, and reproducibility of the real-time HCV RNA quantitation which allows the screening of large numbers of samples, combined with its wide dynamic range, make this method especially suitable for monitoring of the viral load during therapy and tailoring of treatment schedules.

Adult↗
PubMed1999

Evaluation of soft-tissue masses using segmented color Doppler velocity images: preliminary observations.

OBJECTIVE: We report our initial experience with segmented color Doppler velocity-based estimates of tumor vascularity for various histogically proven soft-tissue masses. SUBJECTS AND METHODS: Color Doppler sonography of 23 histologically proven masses in 22 patients was performed. Digital color Doppler images were acquired directly off the scanner output or from video recordings and stored on a personal computer as 24-bit gray-scale and color composite images. A color Doppler velocity segmentation and analysis algorithm was applied to the digital images, from which we calculated the normalized percentage of color Doppler area. Normalization was determined by expressing color Doppler area as a percentage of the area enclosed by a preselected region of interest. We also calculated mean percentage, SD, and cumulative distribution of color Doppler area, relative to a fixed threshold, for the acquired image data sets. RESULTS: Estimates of mean percentage of color Doppler area showed a dynamic range of at least two or three orders of magnitude between lowest and highest values obtained. A scatterplot of mean percentage of color Doppler area versus SD of percentage of color Doppler area showed a linear monotonic relationship (r2 = .92), illustrating increasing vascular heterogeneity with mean vascularity. Preliminary data also suggest the presence of at least two distinct groups of masses (p < .0001) based on these vascularity estimates. One group corresponds to high-grade lesions in which tumor angiogenesis is expected to be important in predicting biologic behavior. The second group appeared to have little or no relationship to tumor vascularity or was of an intermediate (or lower) histologic grade. CONCLUSION: Quantitative color Doppler estimates of tumor vascularity can be obtained over a wide dynamic range. Such estimates provide a mechanism to assess vascular heterogeneity of soft-tissue tumors. Preliminary data suggest that two biologically distinct groups of masses may be separable on the basis of quantitative velocity-based estimates of tumor vascularity as obtained from color Doppler sonography.

Adolescent↗
PubMed2002

Quality rating test of hearing aid benefit in the NIDCD/VA Clinical Trial.

OBJECTIVE: As part of a large clinical trial that compared three hearing aid circuits using several evaluation methods, judgments about quality of listening experiences were sought from all subjects. Three dimensions were examined: loudness, noise interference and overall liking (quality). DESIGN: Eight Audiology units in VA Medical Centers participated. Three hearing aid circuits were compared: linear peak clipper, compression limiter, and wide dynamic range compressor. The experimental design was a three-period, three-treatment crossover design. Baseline measures were made using a battery of tests in unaided conditions. Subjects (N = 360) were then stratified by participating site and randomized to one of six sequences of the three hearing aid circuits. Each circuit was fit binaurally and all subjects used each of the three circuits for 3 mo. All outcome measures were administered in unaided and aided conditions after each 3-mo period. The study used a double-blind strategy, i.e., neither the audiologist giving the tests nor the subject knew which circuit was being used. A different audiologist programmed the devices. RESULTS: For loudness judgments, soft and loud presentations of speech in quiet and in babble competition were judged more comfortable via the wide dynamic range circuit. The noise interference tasks and overall liking of the listening experience showed few significant differences across circuits. All circuits made the listening experience more comfortably loud for soft and conversation-level speech. CONCLUSIONS: Differences across circuits in terms of the overall quality of the listening experience and how noise interference was rated were small. Only isolated conditions, usually favoring the WDRC circuit, reached significance levels. The loudness dimension results were clearer. The WDRC circuit made sounds at either the loud or soft extreme more comfortable. When subjects were grouped by amount and configuration of hearing loss, the advantages for the WDRC and to a lesser extent the linear compression-limited circuit were clearest among subjects with mild hearing losses with a >10 dB/octave high-frequency drop, and those with moderate, relatively flat hearing losses.

Aged↗
PubMed2002

Detection and quantification of insertion/deletion variations by allele-specific real-time PCR: application for genotyping and chimerism analysis.

The DNA-based quantitative analysis of genetic chimerism is becoming increasingly more important for molecular biology in general and molecular medicine in particular. Useful genomic targets for these analyses are polymorphic sequences, but here the problem of a reliable quantification with high dynamic range is not yet satisfactorily solved. To this end we have combined the allele-specific amplification with a real-time PCR-based quantification for rapid allelotyping and chimerism analysis. The sequence variations are discriminated by the 3'-end of the allele-specific primer. Amplification is monitored by SYBR-Green I fluorescence. We demonstrate the efficiency of this method for two clinically relevant targets: (i) the 10 bp insertion/deletion polymorphism in the promoter of the factor VIIc (F-VIIc) gene and (ii) the 4G/5G single nucleotide polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene. Both polymorphisms are associated with clinical risk factors. Allelotyping results were in complete agreement with those obtained by reference methods. Mixed chimeric DNA samples could be quantified reliably with a dynamic range of 1:3000 for an easy target (F-VIIc) and of 1:64 for a difficult target (PAI-1). Our protocol is particularly useful for rapid, reliable and inexpensive genotyping and quantitative chimerism analysis without requiring expensive fluorophor dye labelled probes.

Alleles↗
PubMed2000

Intrathecally administered endotoxin or cytokines produce allodynia, hyperalgesia and changes in spinal cord neuronal responses to nociceptive stimuli in the rat.

Inflammatory processes occurring within the central nervous system (CNS) can produce 'illness induced behaviours' which include fever, sleep and the development of allodynia and hyperalgesia. Here we demonstrate the effects of the pro-inflammatory mediators, bacterial endotoxin, and rat recombinant interleukin 1 beta (rrIL-1 beta) or tumour necrosis factor-alpha (rrTNF alpha) on the integration of somatosensory information at the single neuronal level, via recordings from wide-dynamic range neurones in the dorsal horn of the spinal cord in anaesthetized rats. Intrathecal administration of E. coli lipopolysaccharide (LPS, 10 and 100 microg, i.t.) enhanced the activity of dorsal horn neurones, including facilitation of neuronal post-discharge. Intrathecal administration of IL-1 beta (5-5000 pg) or TNF-alpha (5-5000 pg) enhanced dorsal horn neuronal responses, including the acute responses to C-fibre stimulation, wind-up and post-discharge, however, the effects of IL-1 beta were more robust than those of TNF alpha. Intrathecal administration of IL-1 beta (1-1000 pg) also leads to the development of mechanical allodynia and hyperalgesia. On the other hand intrathecal application of TNF alpha did not produce changes in sensitivity to mechanical stimuli. Changes in the activity of spinal wide-dynamic range neurones induced by local inflammation may provide a pathomechanism for the clinical pathology of central pain syndrome, which can accompany CNS disease or acute CNS injury.

Animals↗
PubMed1981

Rate-versus-intensity functions and related AP responses in normal and pathological guinea pig and human cochleas.

Cochlear fiber discharge rate-versus-intensity functions, across frequency, have been measured in pathological guinea pig cochleas (ototoxic antibiotic poisoning) and compared with the normal animal. The frequency dependence of the slopes of these functions is reduced in cochlear pathology which results in minimum threshold elevations of more than 50 dB, i.e., there is a reduction in this frequency dependent nonlinearity. The rate functions at characteristic frequency (CF) become abnormally steep (e.g., 4--5 spikes/s/dB compared with a normal 1--2 spikes/s/dB), and comparable to those of the low-frequency tail region of normal cochlear fibers. The CF dynamic range is reduced from 30--40 to 10--15 dB in cochlear pathology. The fiber study has been confirmed by (and has confirmed) a method of indirect measurement of rate functions from AP suppression-versus-intensity functions using a pure-tone forward masking paradigm [Abbas and Gorga, J. Acoust. Soc. Am. 69, 492--499 (1981)]. This method has been used in normal and pathological guinea pigs, and the results parallel the single fiber study. In addition, AP suppression functions, across frequency, have been obtained in human subjects with (near) normal hearing thresholds, and in patients with sensorineural hearing loss of cochlear origin, during transtympanic electrocochleography. The AP suppression curves in pathology indicate, as for the animal studies, a loss of the frequency dependency of the rate function slopes, and predict steep rate functions at CF (and thus reduced dynamic range) in cochlear deafness. The findings are related to loudness recruitment.

Acoustics↗
PubMed2006

Performance of the automated Abbott RealTime HIV-1 assay on a genetically diverse panel of specimens from London: comparison to VERSANT HIV-1 RNA 3.0, AMPLICOR HIV-1 MONITOR v1.5, and LCx HIV RNA Quantitative assays.

Automated RNA extraction and quantitation of HIV-1 by real-time PCR offer potential advantages of efficient sample processing, improved sensitivity, expanded dynamic range and reduced contamination risk. In this study, plasma was collected from 100 HIV-1 infected patients visiting The Courtyard Clinic of St. George's Hospital in London, United Kingdom (UK). Viral loads measured using the automated Abbott RealTime HIV-1 assay (m2000sp sample preparation and m2000rt amplification and detection instruments) were compared to results obtained with Versant HIV-1 RNA 3.0 (bDNA), AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5) and LCx HIV RNA Quantitative (LCx HIV) assays. Based on gag p24, pol integrase, and env gp41 sequences, the panel included 26 subtype A, 20 B, 27 C, 10 D, 1 CRF01_AE, 3 CRF02_AG and 13 recombinant viruses. RealTime HIV-1, bDNA, Monitor v1.5 and LCx HIV quantitated 82, 74, 82, and 83% of samples, respectively, with 82, 71, 69 and 80 of the 100 samples measured within the dynamic ranges. Viral loads were highly correlated with 99% of values within 1 log(10) copies/ml between tests. The automated m2000 system and RealTime HIV-1 assay can increase laboratory throughput, enhance overall efficiency and reduce operator-associated errors while providing reliable quantitation of genetically diverse strains of HIV-1.

Automation↗
PubMed2003

Development and validation of a real-time PCR assay for the detection and quantitation of p53 recombinant adenovirus in clinical samples from patients treated with Ad5CMV-p53 (INGN 201).

The purpose of this study was to assess the usefulness of real-time PCR as a quantitative, highly reproducible, and sensitive method, for detecting and quantifying p53 recombinant adenovirus in biological samples from cancer patients receiving injections of Ad5CMV-p53. The dynamic range of this real-time PCR-based assay was wide (at least five orders of magnitude). Our assay used an internal positive control in the same PCR tube that is capable of detecting residual PCR inhibitors. Serial spiked samples in plasma with known quantities of Ad5CMV-p53 were evaluated. The minimum detection limit was 2 pfu per PCR (approximately 50 pfu per ml of plasma) and the quantification values were reproducible. A total of 2069 controls tested with 1780 plasma samples from 286 patients enrolled in gene therapy trials using Ad5CMV-p53 were investigated. Using calibrators to adjust the quantitation value, the results confirmed the good performance of the assay. In conclusion, the high sensitivity, simplicity and reproducibility of the real-time Ad5CMV-p53 assay, allowing screening of large numbers of samples, combined with its wide dynamic range, make this method particularly suitable for monitoring gene therapy trials.

Adenoviruses, Human↗
PubMed2001

Applications of new liquid chromatography-tandem mass spectrometry technologies for drug development support.

We have evaluated (i) a multiplexed electrospray interface, (ii) serial sample introduction, and (iii) a quadrupole time-of-flight mass spectrometer for quantitative bioanalysis in compliance with good laboratory practice. These evaluations were done using a 96-well plate liquid chromatography-tandem mass spectrometry method for the quantitation of loratadine and its metabolite, descarboethoxyloratadine. The assay has a dynamic range of 1-1000 ng/ml with 5.56 pg of each analyte being injected on-column at the limit of quantitation. For the four-channel multiplexed electrospray experiments, one-run validations were performed simultaneously in rat, rabbit, mouse and dog plasma. In the four-stream serial experiments, the total run time of the assay was reduced from 3.5 to 0.35 min, resulting in a net acquisition time of 11 s. Four simulated validation runs with standard and quality control solutions were analyzed. Precision and accuracy for standards and quality control samples met US Food and Drug Administration recommended criteria for both the drug and the metabolite using those two approaches. In addition, a quadrupole time-of-flight mass spectrometer was used as a detector in the tandem mass spectrometry mode for the loratadine assay. Our results demonstrated that a dynamic range of three orders of magnitude could be achieved using the quadrupole time-of-flight mass spectrometer, making it useful for quantitation in preclinical toxicology studies.

Animals↗
PubMed1982

Serum morphine levels in cats during dorsal horn neuron suppression by spinally administered morphine.

The degree of serum uptake of morphine following spinal morphine administration was measured in cats. Total serum morphine levels (free plus conjugated) were determined during suppression of noxiously evoked wide dynamic range neuron activity by spinally administered morphine and compared with total serum morphine levels following i.v. administration of similar doses. The serum levels at 30 minutes after spinal application of morphine (a time at which there was significant suppression of noxiously evoked neuronal activity) were low; after 0.1 and 0.25 mg the levels were 6.5 (n=6) and 12.5 (n=5) ng/ml respectively. In contrast, intravenous administration of the same doses (0.1 and 0.25 mg) produced levels at 30 minutes of 24 and 36 ng/ml respectively, while an intravenous dose commonly used in earlier neurophysiologic studies (2 mg/kg) produced serum levels in excess of 400 ng/ml. These results indicate that although there is systemic uptake following spinal administration of morphine, the serum levels achieved are much lower than those following intravenous administration of a comparable dose, and are insufficient to explain the resultant suppression of wide dynamic range neurons.

Animals↗
PubMed2004

A high-performance liquid chromatographic-atmospheric pressure chemical ionization-tandem mass spectrometric method for determination of risperidone and 9-hydroxyrisperidone in human plasma.

Risperidone, a benzisoxazole derivative, is an antipsychotic agent used for the treatment of schizophrenia. We developed a liquid chromatographic-atmospheric pressure chemical ionization-tandem mass spectrometric (LC-APCI-MS-MS) method with improved sensitivity, selectivity, and dynamic range for determination of risperidone and 9-hydroxyrisperidone in human plasma. A structural analogue of risperidone, RO68808 (5 ng/mL), is added as the internal standard to 1 mL of human plasma. Plasma is made basic, extracted with pentane/methylene chloride (3:1), the organic phase evaporated to dryness, and the residue is reconstituted in water with 0.1% formic acid/acetonitrile (20:1). For LC-MS-MS analysis, a Metachem Inertsel HPLC column (2.1 x 150 mm, 5-microm particle size) is connected to a Finnigan TSQ7000 tandem MS via the Finnigan API interface. Both electrospray (ESI) and APCI produced predominantly MH(+) ions for the two analytes and the internal standard. Ions detected by selected reaction monitoring correspond to the following transitions: m/z 411 to 191 for risperidone, m/z 427 to 207 for 9-hydroxyrisperidone, and m/z 421 to 201 for the internal standard. APCI provided a larger dynamic range (0.1 to 25 ng/mL) and better precision and accuracy than ESI. Intrarun accuracy and precision determined at 0.1, 0.25, 2.5, and 15 ng/mL were within 12% of target with %CVs not exceeding 10.9%. Interrun accuracy and precision determined at the same concentrations were within 9.6% of target with %CVs not exceeding 6.7%. Analytes were stable in plasma after 24 h at room temperature, 2 freeze-thaw cycles, and 490 days at -20 degrees C.

Antipsychotic Agents↗
PubMed2005

Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples.

The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log(10) was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.

Affinity Labels↗
PubMed1990

Immunometric assay by flow cytometry using mixtures of two particle types of different affinity.

An improved dynamic range in a particle based flow cytometric immunoassay for carcinoembryonic antigen (CEA) was obtained using a binary mixture of two distinguishable particle types, namely particles of 7 and 10 microns diameter that were distinguishable by their light scattering characteristics in the flow cytometer. The two particle types were coated with antibody of the same specificity but different affinity. The association constants were 3.2 x 10(10) and 3.3 x 10(9) for the antibodies on the 7 and 10 micron particles, respectively. A dilution series of CEA samples was incubated with aliquots of the particle mixture and secondary biotin-streptavidin-phycoerythrin-conjugated antibody directed against a different epitope on the CEA molecule. The fluorescence intensity of the two particle types was measured flow cytometrically, and a double standard curve plotted from the mean logarithmic fluorescence values. The precision profile derived from the standard curve demonstrated that an increase in the dynamic range of about 50% (from 2 to 3 log) was obtained by using a mixture of high and low affinity particles, compared to using the high affinity particles alone.

Antibodies, Monoclonal↗
PubMed1997

Auditory stimulus intensity and reaction time in listeners with longstanding sensorineural hearing loss.

OBJECTIVE: This study examined relationships among sound level, subjective loudness, and reaction time in listeners with longstanding sensorineural hearing loss and loudness recruitment. DESIGN: A simple reaction-time test was performed by 10 hearing-impaired (HI) subjects with varying degrees of recruitment and by 10 normal-hearing (NH) control subjects. Both groups listened to 0.5 kHz tones presented at a soft level, representing the soft endpoint of a subject's functional dynamic range, and a loud level representing the loud endpoint. In one condition, the loudness level of stimuli within a block was fixed, and hence predictable; in another it varied randomly within a block between loud and soft, adding uncertainty to the simple reaction time task. A test with exactly the same design but using visual stimuli was also performed. RESULTS: 1) In general, the HI subjects responded to auditory stimuli with normal or near-normal mean reaction times when stimulus loudness was predictable. 2) Introduction of uncertainty disrupted the reaction time performance of some, but not all, of the HI subjects. 3) Both the HI and the NH subjects responded more quickly to loud tones than to soft ones. Under the predictable loudness condition the magnitude of this "speed up" was nearly identical for the two groups despite the fact that the HI group had, on average, only half the physical dynamic range of the NH group. 4) Visual reaction-time performance was equivalent between the HI and NH groups in all important respects. CONCLUSIONS: Despite long-term auditory deficits, HI subjects' ability to respond quickly to simple auditory signals is not substantially impaired, particularly when listening under predictable loudness conditions. Although physical soft-loud ranges will generally be narrower than normal for HI subjects, their reaction time performance at the endpoints of that range is likely to be near normal.

Adult↗
PubMed2006

A large field CCD system for quantitative imaging of microarrays.

We describe a charge-coupled device (CCD) imaging system for microarrays capable of acquiring quantitative, high dynamic range images of very large fields. Illumination is supplied by an arc lamp, and filters are used to define excitation and emission bands. The system is linear down to fluorochrome densities <<1 molecule/microm2. The ratios of the illumination intensity distributions for all excitation wavelengths have a maximum deviation approximately +/-4% over the object field, so that images can be analyzed without computational corrections for the illumination pattern unless higher accuracy is desired. Custom designed detection optics produce achromatic images of the spectral region from approximately 450 to approximately 750 nm. Acquisition of a series of images of multiple fluorochromes from multiple arrays occurs under computer control. The version of the system described in detail provides images of 20 mm square areas using a 27 mm square, 2K x 2K pixel, cooled CCD chip with a well depth of approximately 10(5) electrons, and provides ratio measurements accurate to a few percent over a dynamic range in intensity >1000. Resolution referred to the sample is 10 microm, sufficient for obtaining quantitative multicolor images from >30,000 array elements in an 18 mm x 18 mm square.

DNA↗
PubMed2003

Determining optimal gel sensitivity in optical CT scanning of gel dosimeters.

A method for determining the gel sensitivity that is necessary for obtaining optimal image contrast in optical CT scanning of gel dosimeters is presented. The effective dynamic range of the OCTOPUS-ONE research scanner (MGS Research, Inc., Madison, CT) is analyzed. Optical density increments for selected straight-line paths across a gel cylinder to be scanned are calculated based on the optical properties of the polymer gel and the dose distribution from a commercial treatment planning system (Cadplan, Varian Corporation, Palo Alto, CA). Maximum optical density increment across the entire gel is obtained by searching the gel cylinder over a set of transverse planes at different rotational angles. The application of this quantity as a criterion for optimizing the quality of the optical CT scanning is demonstrated through dose verification of two representative treatment plans. When the MU dependence of the dose distribution for a treatment plan is linear, as is the case for static field irradiation, it is possible to scale the treatment plan such that the intensity variation of the signals received by the photodetector spans its entire dynamic range. For treatment plans that are possibly nonlinear, IMRT plans, for example, modification of the sensitivity of the gel material is necessary for the high-dose signals to be collected at a certain signal-to-noise ratio. Results obtained using the optimized CT scanning approach are compared with those from the treatment planning system and the film measurement.

Algorithms↗
PubMed2002

Development and evaluation of an internally controlled semiautomated PCR assay for quantification of cell-free cytomegalovirus.

Quantification of circulating human cytomegalovirus (HCMV) is useful in clinical contexts such as virological surveillance of bone marrow transplant recipients and monitoring of antiviral therapy. This report describes an internally controlled, quantitative, semiautomated, HCMV genome assay that was developed primarily to measure HCMV DNA in the plasma of severely leucopaenic patients. It exhibits greater sensitivity, wider dynamic range and higher sample throughput than a number of previously described commercial and "in-house" assays. Viral DNA extraction from EDTA plasma samples was automated using a BioRobot 9604 (Qiagen). HCMV strain AD169 was used to prepare a calibration curve and murine cytomegalovirus (MCMV) strain Smith was added as internal control to all calibration standards and test samples. Amplification was performed using a set of primers based on the HCMV UL50 region, capable of amplifying both human and murine CMV. The yield of biotinylated polymerase chain reaction (PCR) products was estimated using HCMV-specific and MCMV-specific enzyme-labelled probes and automated chemiluminescence detection. Log-transformed HCMV-to-MCMV signal ratios were calculated and used for quantification of test samples against simultaneously extracted MCMV-spiked calibration standards. Evaluation of the assay sensitivity by Probit analysis demonstrated a 95% probability of detection at 100 HCMV genomes per ml of plasma; the dynamic range was shown to be > or = 4 log(10). A total of 315 samples from 61 bone marrow transplant patients were analysed by both the quantitative PCR (qPCR) and by a previously validated nested nonquantitative PCR (NQPCR). A high level of concordance (90%) was observed between the two assays, although the qPCR assay exhibited slightly greater sensitivity.

Base Sequence↗
PubMed2000

Glycinergic and GABAergic control of intensity-response function of frog ERG waves under different conditions of light stimulation.

The effect of glycinergic blockade by strychnine and GABAergic blockade by picrotoxin on the intensity-response function and time course of ERG b- and d-wave was investigated in dark and light adapted frog eyecups as well as in chromatically adapted eyecups, in which the responses were predominantly mediated by one photoreceptor type. Both of the blockers markedly increased the maximal response amplitude and thus increased the contrast gain of the mechanisms generating ERG waves. Strychnine but not picrotoxin narrowed the dynamic range of the b-wave in all eyecups. The glycine and GABAergic control on retinal sensitivity however, depended on photoreceptor input. The b-wave sensitivity was increased to the greatest extent when green rods mediated the response; it was slightly increased when the response was mediated by red cones and was not changed at all when red rods mediated the b-wave. On the other hand the d-wave sensitivity was enhanced regardless of photoreceptor input so that it became equal (dark) or even greater (light adapted eyes) than that of the b-wave. Strychnine shortened but picrotoxin increased the implicit time of the b-wave and both the blockers markedly slowed the d-wave time course. Thus the blockers considerably diminished or fully eliminated the initial difference in time course between the ON and OFF response. The glycine- and GABAergic blockade did not principally alter the light adaptation process expressed in decreasing retinal sensitivity, narrowing the dynamic range and speeding the time course of the responses with increasing background illumination.

Animals↗