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PubMed2005

Robust quantification of in vitro angiogenesis through image analysis.

An automated image analysis method for quantification of in vitro angiogenesis is presented. The method is designed for in vitro angiogenesis assays that are based on co-culturing endothelial cells with fibroblasts. Such assays are used in many current studies in which anti-angiogenic agents for the treatment of cancer are being sought. This search requires accurate quantification of the stimulatory and inhibitory effects of the different agents. The quantification method gives lengths and sizes of the tubule complexes as well as the numbers of junctions in each of them. The method is tested with a set of test images obtained with a commercially available in vitro angiogenesis assay. The results correctly indicate the inhibitory effect of suramin and the stimulatory effect of vascular endothelial growth factor. Moreover, the image analysis method is shown to be robust against variations in illumination. We have implemented a software package that utilizes the methods. The software as well as a set of test images are available at http://www.cs.tut.fi/sgn/csb/angioquant/.

Algorithms↗
PubMed2001

Examination of in vitro chemosensitivity test using collagen gel droplet culture method with colorimetric endpoint quantification.

To develop a simpler method of performing the collagen gel droplet-embedded culture drug sensitivity test (CD-DST), we examined the introduction of colorimetric quantitative determination of images for evaluation of anticancer effect against cancer cells alone in the presence of fibroblasts, based on differences in proliferative morphology and stainability with neutral red of cells within collagen gel drops determined using a video-microscope and NIH Image software. In examinations using a human cancer cell line and a fibroblast cell line, a high degree of linearity between number of cancer cells and image-optical density was found within the range of 10(2) - 10( 6) cells / droplet (r (2) = 0.933). Using NIH Image, fibroblast cells could be eliminated at a cut-off value of 128, and an immunocytochemical method demonstrated that the cells eliminated from the image were indeed fibroblasts, and those remaining were cancer cells. CD-DST was carried out with mixtures of cancer cells with fibroblasts at various ratios, and the feasibility of evaluating anticancer activity in cancer cells alone with no effect of fibroblasts at any mixing ratio was confirmed. In addition, for CD-DST of primary cell cultures of human lung cancers collected at the time of surgery, a high correlation between results obtained with the volume supplementation method, a current cell quantification method, and those with the imaging colorimetric quantification method was obtained (r = 0.933). These results indicate that introduction of imaging colorimetric quantification utilizing NIH Image makes CD-DST a quick and simple method that should be highly useful for clinical chemosensitivity testing using primary cell cultures of human cancers.

Antineoplastic Agents↗
PubMed1995

Quantification of androgen receptor and follicle-stimulating hormone receptor mRNA levels in human and monkey testes by a ribonuclease-protection assay.

A sensitive, solution-hybridization ribonuclease-protection assay (RPA) was established to quantify the expression of mRNA for the androgen receptor (AR) and follicle-stimulating hormone receptor (FSHR) in total RNA samples isolated from tissues of the cynomolgous monkey, human testes obtained from elderly patients undergoing orchidectomy because of prostatic carcinoma or from transsexual men undergoing gender reassignment as well as human cell lines DU 145, REP and RVP. Sensitivity experiments revealed that, in the human and monkey, 1-2 micrograms of total RNA were sufficient to achieve quantifiable signals of the different receptor mRNA species. Quantification of AR and FSHR mRNA levels showed a 1.7-fold higher expression of AR mRNA and a 2.4-fold higher expression of FSHR mRNA in the monkey testes compared to human testes from patients with prostatic carcinoma. Normal spermatogenesis in both human and monkey testes indicated no relationship between spermatogenic status and receptor expression. The significantly lower expression of AR and FSHR mRNA in humans than in monkeys might therefore be either age- or species-related. Quantification of mRNA for AR and FSHR in the testis of the transsexual patients undergoing oestrogen and antiandrogen treatment displayed a drastic increase (4.5-fold) in mRNA for the AR, whereas mRNA for the FSHR was barely detectable. Due to its high sensitivity, reproducibility and its ability to quantify mRNA transcripts, the RPA is a useful tool for investigating expression of low abundant receptor genes and their regulation when only very small amounts of tissue are available. Furthermore, it is suitable for use in clinical and experimental studies in which accurate quantification of transcripts is necessary.

Aged↗
PubMed1990

A comparison of three quantification methods for estimation of respiratory sinus arrhythmia.

The empirical literature has shown that respiratory sinus arrhythmia is a sensitive noninvasive index of parasympathetic cardiac control. Nevertheless there has been no general agreement among investigators as to the most preferable quantification technique for assessing respiratory sinus arrhythmia, although there has been much speculation that specific estimation techniques are more or less reflective of vagal processes and could be more or less contaminated by other influences upon heart period variability unrelated to respiration. This study compared three quantification procedures for estimating respiratory sinus arrhythmia (RSA): (1) a spectral analytic technique, (2) a complex detrending approach removing periodic and aperiodic cardiac variations unrelated to respiration, and (3) a time-domain, peak-valley procedure employing inspiratory and expiratory periods as windows for determining range of cardiac-interval fluctuations associated with respiratory phase. Measures derived from these techniques were intra- and interindividually compared using three different samples of male subjects, including students, adult normotensives, and adult hypertensives. All interindividual correlations between measures yielded coefficients above .92 and the mean within-subject correlation across 42 individuals was .96, thus indicating a marked degree of comparability between measures. Additionally, given that much evidence indicates lawful within-individual relations between RSA amplitude and respiratory parameters, we employed respiratory period as an external criterion and compared intraindividual correlations between this variable and (2) and (3); results showed that (3) was significantly more highly associated with respiration than was (2), although the mean r's for the two measures did not diverge greatly (.91 vs. .84). Finally, inspection of the data and further regression analyses did not suggest that any of the RSA estimates were differentially contaminated by other components of cardiac variability. Our findings suggest that the three techniques are almost equivalent as indices of cardiac vagal tone and would appear to ease concerns about the inferiority of any of the procedures. Choice of a quantification procedure should therefore be tailored to the specific empirical needs of an investigation. The advantages and disadvantages of each method are discussed.

Adult↗
PubMed2005

Quantification of Escherichia coli by kinetic 5'-nuclease polymerase chain reaction (real-time PCR) oriented to sfmD gene.

AIMS: A kinetic 5'-nuclease polymerase chain reaction (real-time PCR) for the quantification of Escherichia coli was developed. METHODS AND RESULTS: Specific primers and a fluorogenic probe oriented to sfmD gene, encoding a putative outer membrane export usher protein, were designed. The PCR system was highly specific and sensitive for E. coli, as determined with 37 non-E. coli strains (exclusivity, 100%) and 24 E. coli strains (inclusivity, 100%). When used in real-time PCR, linear calibration lines were obtained in the range from 10(2) to 10(8) CFU ml(-1) for three E. coli strains. Salmonella Enteritidis (10(6) CFU ml(-1)) or Citrobacter freundii (10(6) CFU ml(1)) had no effect on quantification of E. coli by the method. CONCLUSIONS: The developed real-time PCR is suitable for rapid quantification of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications.

Bacterial Outer Membrane Proteins↗
PubMed2005

Advances in digital quantification technique enhance discrimination between mild and advanced liver fibrosis in chronic hepatitis C.

BACKGROUND AND AIMS: The necessity of liver biopsy for staging fibrosis and its quantification in patients with chronic hepatitis C (CHC) remains controversial. Semiquantitative scoring of fibrosis is considered more subjective and less objective than digital quantification by image analysis. However, measurement of fibrosis using digital image analysis is thought to be less reliable in determining early stage fibrosis as compared with advanced fibrosis or cirrhosis. Our aims were to correlate all Ishak stages of fibrosis (0-6) with fibrosis percentage (%) using computerized digital image analysis, and thereby seek to improve discrimination between varying levels of liver fibrosis. METHODS: Fibrosis % data were obtained by image analysis on 164 trichrome-stained liver biopsies from untreated patients with CHC, representing all Ishak stages of fibrosis. RESULTS: Digital analysis of fibrosis % was highly correlated with Ishak scores of fibrosis (Kendall's tau-beta=0.86, P<0.001). Receiver-operator characteristic curves showed reliable discriminative capability of our digital image measurement of fibrosis when compared with semiquantitative assessments of fibrosis. Excellent interobserver reliability was found. CONCLUSIONS: Recent advances in digital quantification of fibrosis have resulted in improved discrimination between the varying stages of liver fibrosis, including mild fibrosis. This method is reproducible, can detect early as well as advanced fibrosis or cirrhosis, may prove to be the best assessment of mild fibrosis, and may be more precise than semiquantitative estimation of changes for monitoring fibrosis progression or regression during clinical therapeutic trials.

Azo Compounds↗
PubMed2004

Critical steps in electronic volume quantification of gingival crevicular fluid: the potential impact of evaporation, fluid retention, local conditions and repeated measurements.

BACKGROUND AND OBJECTIVES: Various methodological factors may operate during clinical gingival crevicular fluid (GCF) sampling, volume quantification or subsequent laboratory analysis. For precise volume quantification, specific concern for generation and maintenance of a reliable calibration curve, the potential risk of GCF loss as a result of evaporation or fluid retention on actual volume and the impact of local conditions is needed because each of these factors may act as a source of subsequent volumetric distortions. Thus, the present study aimed to analyse the impact of sample transfer time on the rate of evaporation and the possibility of fluid retention, and the impact of local conditions and number of replicated measurements on the reliability of calibration data. MATERIALS AND METHODS: To analyse evaporative errors, standardized Periopaper strips provided with known test volumes (0.1 microl, 0.2 microl, 0.5 microl and 0.6 microl) were transferred to Periotron 8000 with different time intervals (immediately, 5 s, 30 s and 60 s). For fluid retention, after quantifying the actual volume of the strips provided with known volumes (0.1 microl and 0.6 microl) of two test fluids, a second set of measurements was performed using dry strips. To determine the impact of local conditions (temperature and humidity) and the validity of 3, 5 and 20 replications (0.0-0.6 microl with 0.1- microl increments) on device calibration for 20 degrees C and 25 degrees C, electronic readings were obtained from three devices at three different locations. Differences in volumetric data in each experimental design were statistically analysed. RESULTS: No significant fluid loss was observed within 5 s, but evaporation clearly led to volumetric distortions with extending transfer times (30 s or 60 s) (p < 0.05). Measurable amounts of fluid retention were found for both volumes and both test fluids, but not with identical patterns. Local conditions resulted in unique calibration data for each test volume and for each device. Although a 5 degrees C increase generally provided higher readings, this was not observed for all devices at all volumes. Additional replicates (n = 5 or n = 20) did not seem to add any further reliability to the triplicate scores for the given test volumes. CONCLUSION: The findings of the present study confirm the reliability of triplicate readings, and uniqueness of each device and electronic data and the distinct impact of local environmental conditions on the generation/maintenance of calibration scores for each particular device. Furthermore, they underline time-dependent evaporation and fluid retention as additional technical concerns and once again highlight the importance of methodological standardization of the electronic volume quantification process.

Calibration↗
PubMed2006

Novel rinse assay for the quantification of oral neutrophils and the monitoring of chronic periodontal disease.

BACKGROUND AND OBJECTIVES: The aim of this study was to develop a single, rapid, noninvasive oral rinse assay to enable the accurate quantification of oral neutrophils. Products released by neutrophils are partly responsible for the destruction observed in periodontitis. Quantification of oral neutrophil levels is important for understanding their role in periodontal diseases. Previous studies have relied on time-consuming serial rinses and cumbersome counting techniques for the collection and quantification of oral neutrophils. MATERIAL AND METHODS: Patients with chronic periodontal disease provided rinse samples before and after phase I periodontal treatment. Cells in the rinse samples were stained with acridine orange, and neutrophil counts were carried out using a fluorescence microscope and a hemocytometer. RESULTS: This assay allowed us to detect a significant difference in pretreatment oral neutrophil counts between periodontal disease and healthy control groups (p < 0.001). Patients who responded favorably to phase I therapy demonstrated a 43% reduction in oral neutrophil counts compared with their pretreatment levels (p = 0.019). Patients who did not respond to phase I periodontal treatment showed no significant difference in oral neutrophil levels (p = 0.39). CONCLUSION: Oral neutrophil levels, as determined by a rapid oral rinse, reflect the severity of periodontal disease and treatment response. A single, rapid, oral rinse assay is an effective means of collecting and quantifying oral neutrophil levels and may serve as an excellent research tool for further study of the role of neutrophils in periodontal diseases.

Acridine Orange↗
PubMed1997

Three-dimensional ultrasound angiography (power mode) for the quantification of carotid artery atherosclerosis.

Three-dimensional (3D) ultrasound angiography was performed to diagnose carotid artery atherosclerosis. Thirty-five patients (15 women, 20 men) with a history of cerebrovascular disease were examined using conventional color-coded Doppler ultrasound and 3D ultrasound angiography. Carotid stenosis was initially diagnosed using continuous-wave Doppler ultrasound. To determine intraobserver and interobserver reliabilities, 21 patients were evaluated using 3D ultrasound on three occasions. Sixty-five percent of patients were diagnosed with stenosis of more than 50%. Twenty-two percent of plaques had a smooth surface, 72.9% were ulcerated, and 5.1% were indeterminate. Data collection for 3D imaging required 5 minutes per patient, whereas image processing and plaque volume quantification required 30 minutes. Plaque volume ranged from 0.053 to 0.685 ml. The intraobserver and interobserver variabilities were 4.16 and 5.87%, respectively (r = 0.96, p < 0.0001; r = 0.89, p < 0.0001). 3D Color Doppler and 3D ultrasound angiography assessments of plaque volume differed by 8.5%. Plaques were more precisely differentiated using 3D ultrasound, and plaque volume quantification was less affected by echo shadowing after 3D reconstruction. In comparison to other techniques for the quantification of atherosclerotic lesions. 3D ultrasound angiography offers a more precise quantitative method for prospective, clinical studies of atherosclerosis.

Aged↗
PubMed2001

Improved in vitro quantification of the force exerted by the papillary muscle on the left ventricular wall: three-dimensional force vector measurement system.

Recent developments indicate that the forces acting on the papillary muscles can be a measure of the severity of mitral valve regurgitation. Pathological conditions, such as ischemic heart disease, cause changes in the geometry of the left ventricle and the mitral valve annulus, often resulting in displacement of the papillary muscles relative to the annulus. This can lead to increased tension in the chordae tendineae. This increased tension is transferred to the leaflets, and can disturb the coaptation pattern of the mitral valve. The force balance on the individual components governs the function of the mitral valve. The ability to measure changes in the force distribution from normal to pathological conditions may give insight into the mechanisms of mitral valve insufficiency. A unique in vitro model has been developed that allows quantification of the papillary muscle spatial position and quantification of the three-dimensional force vector applied to the left ventricular wall by the papillary muscles. This system allows for the quantification of the global force exerted on the posterior left ventricular wall from the papillary muscles during simulation of normal and diseased conditions.

Animals↗
PubMed2002

Myocardial blood flow quantification with MRI by model-independent deconvolution.

Magnetic resonance (MR) imaging during the first pass of an injected contrast agent has been used to assess myocardial perfusion, but the quantification of blood flow has been generally judged as too complex for its clinical application. This study demonstrates the feasibility of applying model-independent deconvolution to the measured tissue residue curves to quantify myocardial perfusion. Model-independent approaches only require minimal user interaction or expertise in modeling. Monte Carlo simulations were performed with contrast-to-noise ratios typical of MR myocardial perfusion studies to determine the accuracy of the resulting blood flow estimates. With a B-spline representation of the tissue impulse response and Tikhonov regularization, the bias of blood flow estimates obtained by model-independent deconvolution was less than 1% in all cases for peak contrast to noise ratios in the range from 15:1 to 20:1. The relative dispersion of blood flow estimates in Monte Carlo simulations was less than 7%. Comparison of MR blood flow estimates against measurements with radio-isotope labeled microspheres indicated excellent linear correlation (R2 = 0.995, slope: 0.96, intercept: 0.06). It can be concluded from these studies that the application of myocardial blood flow quantification with MRI can be performed with model-independent methods, and this should support a more widespread use of blood flow quantification in the clinical environment.

Animals↗
PubMedDate not supplied

Improved quantification of radionuclide uptake using deconvolution and windowed subtraction techniques for scatter compensation in single photon emission computed tomography.

A comparison of two methods of scatter compensation in single photon emission computed tomography (SPECT) imaging is made on the basis of improvements in quantification. The methods, scatter-window subtraction and constrained deconvolution of an average point source response function (PSRF), are described; the theoretical basis of each method is also briefly assessed. Improvements in relative quantification offered by each method are measured by examining ratios of counts in hot cylinders to counts in a slightly radioactive background. The cylinder/background concentration ratio was varied by a factor of five; the sizes of the cylinders remained constant. Keeping the diameter of the cylinders constant allowed for an assessment of the effect of concentration differences, without the conflicting effect of variation in the size of the hot source. Results showed that while both scatter-window subtraction and constrained deconvolution offer quantification improvements in the final image, the method of prereconstruction deconvolution of a planar PSRF from each planar projection is substantially more successful than either scatter-window subtraction or other methods of implementing the deconvolution procedure.

Biophysical Phenomena↗
PubMed2000

Comparison of methods for quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples.

Two PCR primer sets were developed for the detection and quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples. The specificity and sensitivity of these primers were tested. Both primer sets were suitable for detection, but only one set, cd3F-cd4R, was suitable for the quantification and enumeration of the functional community using most-probable-number PCR and competitive PCR techniques. Quantification of cytochrome cd(1) denitrifiers taken from marine sediment and water samples was achieved using two different molecular techniques which target the nirS gene, and the results were compared to those obtained by using the classical cultivation method. Enumerations using both molecular techniques yielded similar results in seawater and sediment samples. However, both molecular techniques showed 1,000 or 10 times more cytochrome cd(1) denitrifiers in the sediment or water samples, respectively, than were found by use of the conventional cultivation method for counting.

Bacteria↗
PubMed2004

Development and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea.

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the "Epsilonproteobacteria" related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 x 10(3) to 4.4 x 10(9) copies ml(-1) or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 x 10(1) to 2.2 x10(6) copies ml(-1) or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml(-1). The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.

Bacteria↗
PubMed2006

Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis.

Both the viral titer and the genotype significantly determine clinical outcomes and responses to antiviral treatment in chronic hepatitis B virus (HBV) infection. A method was developed for large-scale A-to-G genotyping with simultaneous viral quantification. The assay was run on a LightCycler instrument using hybridization probes. The genotype was determined from the melting points of the probes in a two-step manner. Set 1 amplicons differentiated genotypes B, E, and F from A, C, D, and G and simultaneously quantified viremia by real-time PCR. Melting curve analysis using the set 2-1 amplicon or the set 2-2 amplicon reaction mixture was then used to differentiate these genotype groups into single genotypes. HBV DNA quantification was consistent with that of the Amplicor assay and linear in a range from 10(2) to 10(13) copies/ml. By comparison with the restriction fragment length polymorphism method, 92.3% of 441 samples were accurately genotyped by the current assay. The method should be useful for genotyping and quantification of HBV DNA in areas where all genotypes exist.

DNA Primers↗
PubMed1996

An enhanced-sensitivity branched-DNA assay for quantification of human immunodeficiency virus type 1 RNA in plasma.

The quantification of human immunodeficiency virus type 1 (HIV-1) RNA has facilitated clinical research and expedited the development of antiretroviral drugs. The branched-DNA (bDNA) assay provides a reliable method for the quantification of HIV-1 RNA in human plasma and is considered one of the most reproducible assays ready for use in clinical trials. A series of oligonucleotide probe design and solution changes have been developed to enhance the sensitivity of the bDNA assay while maintaining its performance characteristics. Among the changes incorporated into the enhanced-sensitivity bDNA (ES bDNA) assay to reduce the background level and enhance the signal are the use of shorter overhang sequences of target probes for capture, the cruciform design of target probes for amplification, and the addition of preamplifier molecules. The ES bDNA assay is at least 20-fold more sensitive than the first-generation bDNA assay, yet it maintains a high level of accuracy, linearity, and reproducibility. Further, quantification values obtained with the ES bDNA assay and the first-generation bDNA assay are highly correlated, thus allowing for meaningful comparisons of HIV-1 RNA levels in specimens tested with either assay. The ES bDNA assay may be useful in determining the prognostic value of HIV-1 RNA levels of below 10,000 copies per ml and in assessing the clinical benefit of antiretroviral therapy-induced decreases in plasma HIV-1 RNA sustained at levels of below 10,000 copies per ml.

Anti-HIV Agents↗
PubMed1997

Quantification of human immunodeficiency virus type 1 RNA from dried plasma spots collected on filter paper.

To assess dried plasma spots (DPSs) as a source of material for virus quantification, human immunodeficiency virus type 1 (HIV-1) RNA levels were quantified in matched DPS and liquid plasma samples from 73 infected patients, including 5 neonates and 4 adult patients with acute HIV-1 infection. Quantifications were performed by commercially available assays (NASBA [nucleic acid sequence-based amplification] or Amplicor, or both). There was a strong correlation between HIV-1 RNA levels in plasma and DPSs. More importantly, there was no decline in HIV-1 RNA levels in DPSs stored for as long as 2 weeks at 20 degrees C. Similarly, storage of DPSs for 3 days at 37 degrees C resulted in no decrease in viral RNA levels. For patients with primary infection, the DPS method allowed for the measurement of RNA levels in plasma during the initial spike in the level of viremia and in the subsequent period of suppressed viral replication. DPS quantification was equally informative in the neonatal setting, with all five newborns showing HIV-1 RNA loads of greater than 4.991 log10 copies/ml. We conclude that the viral RNA levels in DPSs are equivalent to those measured in fresh-frozen plasma. The ease and economy of DPS sampling, the minute volumes required, and the unexpected stability of dried RNA suggest that the use of DPSs will be particularly valuable for small-volume neonatal samples and large, population-based studies in which cold storage and transportation present special problems, as is often the case in developing countries. The ability to measure viral changes during primary infection suggests that the method will be useful for assessing vaccine efficacy in large field trials.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Use of multiple competitors for quantification of human immunodeficiency virus type 1 RNA in plasma.

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma has rapidly become an important tool in basic HIV research and in the clinical care of infected individuals. Here, a quantitative HIV assay based on competitive reverse transcription-PCR with multiple competitors was developed. Four RNA competitors containing identical PCR primer binding sequences as the viral HIV-1 RNA target were constructed. One of the PCR primers was fluorescently labeled, which facilitated discrimination between the viral RNA and competitor amplicons by fragment analysis with conventional automated sequencers. The coamplification of known amounts of the RNA competitors provided the means to establish internal calibration curves for the individual reactions resulting in exclusion of tube-to-tube variations. Calibration curves were created from the peak areas, which were proportional to the starting amount of each competitor. The fluorescence detection format was expanded to provide a dynamic range of more than 5 log units. This quantitative assay allowed for reproducible analysis of samples containing as few as 40 viral copies of HIV-1 RNA per reaction. The within- and between-run coefficients of variation were <24% (range, 10 to 24) and <36% (range, 27 to 36), respectively. The high reproducibility (standard deviation, <0.13 log) of the overall procedure for quantification of HIV-1 RNA in plasma, including sample preparation, amplification, and detection variations, allowed reliable detection of a 0.5-log change in RNA viral load. The assay could be a useful tool for monitoring HIV-1 disease progression and antiviral treatment and can easily be adapted to the quantification of other pathogens.

Binding, Competitive↗