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At least 721 records · Page 40Linked to original sources
PubMed1991

Relative quantification of collagen mRNA in fibroblasts by a radioactive polymerase chain reaction technique.

A radioactive polymerase chain reaction (PCR) method has been developed for the relative quantification of the human alpha-2 chain of type I collagen [hu alpha-2(I)] in cells. cDNAs generated by reverse transcription from the total pool of cytoplasmic RNA serve as a template for polymerase chain reaction amplification of a hu alpha-2(I) cDNA primed by two sequence-specific synthetic oligonucleotides. The distinctive 390 bp hu alpha-2(I) cDNA and two Aval fragments of 220 and 170 bp are identified by agarose gel electrophoresis. alpha-32P-dCTP of defined specific activity is included in the PCR reaction and the 390 bp cDNA is excised from the electrophoresis gel to permit direct radioactive quantification of hu alpha-2(I) mRNA. The amount of hu alpha-2(I) mRNA expressed in as few as 111 fibroblasts was determined reliably. In contrast, the hu alpha-2(I) mRNA from at least 5 x 10(5) fibroblasts was required for detection by Northern blot analysis developed with the same cDNA probe radiolabelled with alpha-32P-dCTP by random priming. Human bronchoalveolar lavage (BAL) fluids of six patients with fibrosing lung diseases stimulated the level of expression of hu alpha-2(I) mRNA in cultured human fibroblasts as determined by this technique. The radioactive PCR method thus quantifies hu alpha-2(I) mRNA in fibroblasts with sufficient sensitivity to study fibroblast activation in vitro and detect fibroblast stimuli in human clinical samples.

Autoradiography↗
PubMed1992

Single point quantification of antibody by ELISA without need of a reference curve.

A new method for the quantification of antibodies in the enzyme-linked immunosorbent assay is described. This procedure replaces titer determinations based on end-point dilution of the antibody under investigation. Here, the sample is tested in a single dilution and the optical density (OD) obtained is used in the equation log10 titer = alpha.OD beta. The titer can then be calculated by inserting into the formula the values for the constants alpha and beta, which are specified by the manufacturer for each separate batch of kit reagents. This so-called alpha-method saves time and reagents while providing results which are equal to the titration method in accuracy and superior in precision. The alpha-method is also a simpler and reliable alternative to the use of standard or reference curves for the quantification of the antibodies in I.U./ml.

Antibodies↗
PubMed2005

Nanoscale imaging and quantification of local proteolytic activity.

Proteolytic cleavage of extracellular matrix (ECM) is a critical feature of tumor cell invasion, and affects cancer cell growth, differentiation, apoptosis, and migration. Malignant cells secrete most proteases as inactive proenzymes that undergo proteolytic cleavage for activation, and proteolytic activity is elevated in close proximity to these cells. Therefore, local activity rather than protease concentration determines ECM proteolysis. Precise quantification of local proteolytic activity, functional investigation, and high resolution imaging of morphological ECM alterations have proven difficult. In this study, we present a novel approach for measuring proteolytic activity in the microenvironment of cells by using atomic force microscopy (AFM). Amelanotic melanoma cells (A7-clone) were seeded on fluorescent gelatin or collagen-IV coatings. Proteolysis reduced fluorescence of these coatings. Fluorescence microscopy (FM) in combination with AFM was used to maneuver the AFM-tip to tumor cell induced proteolytic spots. AFM enabled nanoscale volume measurement, three-dimensional reconstruction of single proteins and demonstrated that ECM cleavage is restricted to the proteolytic microenvironment of cancer cells. This method detected significant decreases in molecular weight of protein clusters (-76.6%), matrix volume (-46.6%), and height (-38.1%) between intact and proteolyzed gelatin. Similar parameter changes were demonstrated without FM, by AFM-scanning gelatin in close proximity to invasive cells. Furthermore, AFM depicted significantly stronger local degradation of gelatin than collagen-IV by A7-cells. Taken together, AFM allows specific quantification and imaging of local proteolytic processes at a nanometer level, thus providing a unique method for the functional evaluation of invasiveness and metastatic potential of tumor cells in small scale samples.

Animals↗
PubMed2006

Dynamic-SIMS imaging and quantification of inorganic ions in frozen-hydrated plant samples.

We present here SIMS images of the distribution of inorganic cations (Na, K, Mg and Ca) in frozen-hydrated samples of three plant species, ivy, camomile, and flax. The samples were cryofixed using fast plunge-freezing. Stigmatic images were obtained, at 100 K, under dynamic SIMS conditions by fast atom bombarding (FAB). Even though the images obtained with the frozen-hydrated plant samples are still not of upper quality, they show that the method used to prepare these samples preserves existing ionic gradients between the outer and the inner part of the cells, between adjacent cells, including cells with the same type of differentiation, and between tissues. We also describe the quantification of the relative proportions of the ions in the vacuoles of flax. The reasonable accuracy achieved for quantification of the vacuole ion ratios permitted to show (i) that radial gradients of ion ratios in hypocotyls change when the plant is becoming older and (ii) that large differences may exist between adjacent cortical cells of the same type. The role of these substantial differences in vacuole ion balance ratios is a largely unexplored issue in plant physiology.

Cations↗
PubMed2006

Q-FIHC: quantification of fluorescence immunohistochemistry to analyse p63 isoforms and cell cycle phases in human limbal stem cells.

Fluorescence microscopy has long been used for qualitative characterization of various parameters such as subcellular distribution of proteins, lipids, nucleic acids, and ions. However, quantification of these parameters is complicated by a variety of optical, biological, and physical factors. In the last decade, the progress achieved with powerful softwares and digital image processing systems has facilitated the development of fluorescence immunohistochemistry (FIHC) into a widely used quantitative assay (quantitative-FIHC or Q-FIHC). We describe here a rapid and sensitive Q-FIHC assay based on the use of a laser scanning confocal microscope and advanced image analysis softwares (Zeiss semi automatic LSM 510 and fully automatic Axiovision 4.4) for the detection and quantification of fluorescent intensity in human corneal tissues and cells obtained from small clinical samples. We have used this methodology to characterize and quantify the gene expression profile of p63 and its DeltaNalpha isoform, specific markers of human limbal stem cells. The validity of this method was evaluated through comparative studies with conventional approaches suggesting no significant differences and providing an alternative technique to traditional methods. Since Q-FIHC requires at least 20-fold less cells than traditional techniques, we have adopted it as the main quality control for our limbal cultures destined to clinical application.

Cell Cycle↗
PubMed2002

Reproducibility of in vivo metabolite quantification with proton magnetic resonance spectroscopic imaging.

PURPOSE: To investigate intra- and interscanner in vivo reproducibility of brain metabolite quantification using 1H magnetic resonance spectroscopic imaging (1H-MRSI) (PRESS localization, TE = 30 msec, voxel volume = 2.3 mL) and the linear combination model (LCModel). MATERIALS AND METHODS: One subject had a total of nine scans on three occasions at a single site, and three subjects had single scans at two sites. Coefficients of variation (CVs) were estimated using different statistical models applied to intra- and interscanner data; therefore, only qualitative comparisons may be made between results. RESULTS: CV (intra-/interscanner) for metabolite quantifications were choline, 12.3%/10.1%; creatine, 9.9%/10.6%; glutamate + glutamine, 15.8%/13.6%; myo-inositol, 18.5%/14.7%; and N-acetyl-aspartate + N-acetyl-aspartyl-glutamate, 6.1%/7.0%. Overall, total intra- and intersubject variability was greater than intra- and interscanner variability. CONCLUSION: When quantifying metabolic concentrations using the methods employed in this study, biological factors contribute a greater proportion to measurement variability than measurement errors. Using this technique, intra- and intersite measurement errors are of the same order.

Adult↗
PubMed2001

Aortic and mitral regurgitation: quantification using moving slice velocity mapping.

Comprehensive assessment of the severity of valvular insufficiency includes quantification of regurgitant volumes. Previous methods lack reliable slice positioning with respect to the valve and are prone to velocity offsets due to through-plane motion of the valvular plane of the heart. Recently, the moving slice velocity mapping technique was proposed. In this study, the technique was applied for quantification of mitral and aortic regurgitation. Time-efficient navigator-based respiratory artifact suppression was achieved by implementing a prospective k-space reordering scheme in conjunction with slice position correction. Twelve patients with aortic insufficiency and three patients with mitral insufficiency were studied. Aortic regurgitant volumes were calculated from diastolic velocities mapped with a moving slice 5 mm distal to the aortic valve annulus. Mitral regurgitant flow was indirectly assessed by measuring mitral inflow at the level of the mitral annulus and net aortic outflow. Regurgitant fractions, derived from velocity data corrected for through-plane motion, were compared to data without correction for through-plane motion. In patients with mild and moderate aortic regurgitation, regurgitant fractions differed by 60% and 15%, on average, when comparing corrected and uncorrected data, respectively. Differences in severe aortic regurgitation were less (7%). Due to the large orifice area of the mitral valve, differences were still substantial in moderate-to-severe mitral regurgitation (19%). The moving slice velocity mapping technique was successfully applied in patients with aortic and mitral regurgitation. The importance of correction for valvular through-plane motion is demonstrated.

Adult↗
PubMedDate not supplied

Fuzzy clustering of gradient-echo functional MRI in the human visual cortex. Part II: quantification.

Fuzzy cluster analysis (FCA) is a new exploratory method for analyzing fMRI data. Using simulated functional MRI (fMRI) data, the performance of FCA, as implemented in the software package Evident, was tested and a quantitative comparison with correlation analysis is presented. Furthermore, the fMRI model fit allows separation and quantification of flow and blood oxygen level dependent (BOLD) contributions in the human visual cortex. In gradient-recalled echo fMRI at 1.5 T (TR = 60 ms, TE = 42 ms, radiofrequency excitation flip angle [theta] = 10 degrees-60 degrees) total signal enhancement in the human visual cortex, ie, flow-enhanced BOLD plus inflow contributions, on average varies from 5% to 10% in or close to the visual cortex (average cerebral blood volume [CBV] = 4%) and from 100% to 20% in areas containing medium-sized vessels (ie, average CBV = 12% per voxel), respectively. Inflow enhancement, however, is restricted to intravascular space (= CBV) and increases with increasing radiofrequency (RF) flip angle, whereas BOLD contributions may be obtained from a region up to three times larger and, applying an unspoiled gradient-echo (GRE) sequence, also show a flip angle dependency with a minimum at approximately 30 degrees. This result suggests that a localized hemodynamic response from the microvasculature at 1.5 T may be extracted via fuzzy clustering. In summary, fuzzy clustering of fMRI data, as realized in the Evident software, is a robust and efficient method to (a) separate functional brain activation from noise or other sources resulting in time-dependent signal changes as proven by simulated fMRI data analysis and in vivo data from the visual cortex, and (b) allows separation of different levels of activation even if the temporal pattern is indistinguishable. Combining fuzzy cluster separation of brain activation with appropriate model calculations allows quantification of flow and (flow-enhanced) BOLD contributions in areas with different vascularization.

Adult↗
PubMedDate not supplied

Quantification of mitral regurgitation with MR phase-velocity mapping using a control volume method.

Reliable diagnosis and quantification of mitral regurgitation are important for patient management and for optimizing the time for surgery. Previous methods have often provided suboptimal results. The aim of this in vitro study was to evaluate MR phase-velocity mapping in quantifying the mitral regurgitant volume (MRV) using a control volume (CV) method. A number of contiguous slices were acquired with all three velocity components measured. A CV was then selected, encompassing the regurgitant orifice. Mass conservation dictates that the net inflow into the CV should be equal to the regurgitant flow. Results showed that a CV, the boundary voxels of which excluded the region of flow acceleration and aliasing at the orifice, provided accurate measurements of the regurgitant flow. A smaller CV provided erroneous results because of flow acceleration and velocity aliasing close to the orifice. A large CV generally provided inaccurate results because of reduced velocity sensitivity far from the orifice. Aortic outflow, orifice shape, and valve geometry did not affect the accuracy of the CV measurements. The CV method is a promising approach to the problem of quantification of the MRV.

Blood Flow Velocity↗
PubMed2005

Influence of positional and angular variation of automatically planned short-axis stacks on quantification of left ventricular dimensions and function with cardiovascular magnetic resonance.

PURPOSE: To theoretically and experimentally investigate the influence of the automated cardiovascular magnetic resonance (CMR) scan planning pitfalls, namely inaccurate positioning and tilting of short-axis (SA) imaging planes, on quantification of the left ventricular (LV) dimensions and function. MATERIALS AND METHODS: Eleven healthy subjects and eight patients underwent CMR. Manually and automatically planned SA sets were acquired. To obtain the quantitative measurements of LV function, one observer performed image analysis twice. The agreement between planning methods, as well as the decomposition of the total variation into interstudy and intraobserver components was measured. RESULTS: The decomposition of the total variation showed that the interstudy factor accounts for 70-85% of the total variation, while the rest is due to the intraobserver factor. Moreover, the relative contribution of the interstudy factor remains independent from errors in slice positioning and small angular deviation of SA stacks from the optimal orientation. Good agreement between the theoretical and measured variability factors was observed. CONCLUSION: Global LV function derived from the automatically planned CMR acquisitions yield accurate quantification of the human cardiovascular system. Inaccurate positioning and tilting of SA images does not affect the quantitative measurements of LV function. The computer-aided system for automated CMR has proven clinical applicability.

Adult↗
PubMed2005

Quantification of cerebral perfusion using arterial spin labeling: two-compartment models.

One of the advantages of arterial spin labeling (ASL) techniques over other techniques for measuring cerebral perfusion is that with ASL it is possible to achieve accurate quantification. This is particularly useful in the field of functional imaging, where accurate measurements of perfusion change can help untangle the complex physiological changes that occur following neuronal activation. However, the linearity of the perfusion estimate over a wide range of perfusion values may be more important than absolute values. For several years, single-compartment models have dominated the literature, and it has been assumed that the labeled water diffuses freely throughout the tissue voxel. However, recent work, as summarized in this review, has shown that this assumption is inaccurate and leads to an overestimation of perfusion at low perfusion rates, and an underestimation at high rates. The inclusion of restricted permeability of the capillary wall to water in a two-compartment model offers improved quantification.

Arteries↗
PubMed2006

Accurate quantification of visceral adipose tissue (VAT) using water-saturation MRI and computer segmentation: preliminary results.

PURPOSE: To describe and evaluate the accuracy of water-saturation MRI and a computer segmentation program for quantification of visceral adipose tissue (VAT). MATERIALS AND METHODS: MRI was performed on five patients with whole-volume coverage of the abdomen using two different sequences: 1) a T1-weighted spoiled gradient-echo breath-hold sequence (non-water-saturation) and 2) a T1-weighted spoiled gradient-echo water-saturation breath-hold sequence (water-saturation). The computer segmentation program analyzed the data and calculated VAT volumes (cm3) from both sequences. The data from one patient were additionally processed with the use of a manual technique. The intrastudy reproducibility of the proposed method using the water-saturation MRI sequence and the computer segmentation technique was tested by repeated measures of the automated system analysis (x 10) on MRI data from a single subject to calculate variability. RESULTS: VAT volumes measured by the water-saturation MRI sequences were consistently greater than those measured by the non-water-saturation sequences. Comparison of VAT volumes derived from the water-saturation images and measured by the computer segmentation technique vs. the manual technique showed good correlation (K = 0.8), with a significant time-saving benefit associated with the automated method (5 minutes vs. 1 hour). There was poor correlation between VAT volume measurement calculated by the manual technique and the computer segmentation technique using non-water-saturation images. The reproducibility of the computer segmentation technique using data derived from water-saturation images was high, with a low variability (+/- 5%). CONCLUSION: The results obtained demonstrate that the proposed method may be able to provide accurate quantification of VAT in a highly reproducible and efficient manner.

Body Water↗
PubMed2003

Rapid quantification of HIV protease inhibitors in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

HIV protease inhibitors are important antiretroviral drugs which have substantially reduced the morbidity and mortality associated with HIV-1 infection. Recent data have shown relationships between plasma concentrations of the protease inhibitors and clinical response, which makes therapeutic drug monitoring valuable. We have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), for the routine quantification of the six licensed protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) and the pharmacologically active nelfinavir metabolite M8 in plasma. The sample pretreatment consisted of protein precipitation with a mixture of methanol and acetronitrile using only 100 microl of plasma. Chromatographic separation was performed on an Inertsil ODS3 column (50 x 2.0 mm i.d., particle size 5 microm), with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.5 ml min(-1). The analytical run time was 5.5 min. The use of a 96-well plate autosampler allowed batch sizes up to 150 patient samples. The triple-quadrupole mass spectrometer was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges 0.01-10 microg ml(-1) for indinavir and saquinavir, 0.1-10 microg ml(-1) for amprenavir, 0.05-10 microg ml(-1) for nelfinavir and ritonavir, 0.1-20 microg ml(-1) for lopinavir and 0.01-5 microg ml(-1) for M8. Saquinavir-d(5) and indinavir-d(6) were used as internal standards. The coefficients of variation were always <10% for both intra-day and inter-day precisions for each compound. Mean accuracies were also between the designated limits (+/-15%). The validated concentration ranges proved to be adequate in daily practice. This robust and fast LC/MS/MS assay is now successfully applied for routine therapeutic drug monitoring and pharmacokinetic studies in our hospital.

Chromatography, High Pressure Liquid↗
PubMed2003

Screening for and validated quantification of amphetamines and of amphetamine- and piperazine-derived designer drugs in human blood plasma by gas chromatography/mass spectrometry.

The classical stimulants amphetamine, methamphetamine, ethylamphetamine and the amphetamine-derived designer drugs MDA, MDMA ('ecstasy'), MDEA, BDB and MBDB have been widely abused for a relatively long time. In recent years, a number of newer designer drugs have entered the illicit drug market. 4-Methylthioamphetamine (MTA), p-methoxyamphetamine (PMA) and p-methoxymethamphetamine (PMMA) are also derived from amphetamine. Other designer drugs are derived from piperazine, such as benzylpiperazine (BZP), methylenedioxybenzylpiperazine (MDBP), trifluoromethylphenylpiperazine (TFMPP), m-chlorophenylpiperazine (mCPP) and p-methoxyphenylpiperazine (MeOPP). A number of severe or even fatal intoxications involving these newer substances, especially PMA, have been reported. This paper describes a method for screening for and simultaneous quantification of the above-mentioned compounds and the metabolites p-hydroxyamphetamine and p-hydroxymethamphetamine (pholedrine) in human blood plasma. The analytes were analyzed by gas chromatography/mass spectrometry in the selected-ion monitoring mode after mixed-mode solid-phase extraction (HCX) and derivatization with heptafluorobutyric anhydride. The method was fully validated according to international guidelines. It was linear from 5 to 1000 micro g l(-1) for all analytes. Data for accuracy and precision were within required limits with the exception of those for MDBP. The limit of quantification was 5 micro g l(-1) for all analytes. The applicability of the assay was proven by analysis of authentic plasma samples and of a certified reference sample. This procedure should also be suitable for confirmation of immunoassay results positive for amphetamines and/or designer drugs of the ecstasy type.

Amphetamines↗
PubMed2004

Simultaneous quantification of cyclophosphamide, 4-hydroxycyclophosphamide, N,N',N"-triethylenethiophosphoramide (thiotepa) and N,N',N"-triethylenephosphoramide (tepa) in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

The alkylating agents cyclophosphamide (CP) and N, N', N"-triethylenethiophosphoramide (thiotepa) are often co-administered in high-dose chemotherapy regimens. Since these regimens can be complicated by the occurrence of severe and sometimes life-threatening toxicities, pharmacokinetically guided administration of these compounds, to reduce variability in exposure, may lead to improved tolerability. For rapid dose adaptations during a chemotherapy course, we have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), for the routine quantification of CP, thiotepa and their respective active metabolites 4-hydroxycyclophosphamide (4OHCP) and N, N', N"-triethylenephosphoramide (tepa) in plasma. Because of the instability of 4OHCP in plasma, the compound is derivatized with semicarbazide (SCZ) immediately after sample collection and quantified as 4OHCP-SCZ. Sample pretreatment consisted of protein precipitation with a mixture of methanol and acetronitrile using 100 microl of plasma. Chromatographic separation was performed on an Zorbax Extend C18 column (150 x 2.1 mm i.d., particle size 5 microm), with a quick gradient using 1 mM ammonia solution and acetonitrile, at a flow-rate of 0.4 ml min(-1). The analytical run time was 10 min. The triple quadrupole mass spectrometer was operating in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges 200-40,000 ng ml(-1) for CP, 50-5000 ng ml(-1) for 4OHCP-SCZ and 5-2500 ng ml(-1) for thiotepa and tepa, using 100 microl of human plasma. These dynamic concentration ranges proved to be relevant in daily practice. Hexamethylphosphoramide was used as an internal standard. The coefficients of variation were <12% for both intra-day and inter-day precisions for each compound. Mean accuracies were also between the designated limits (+/- 15%). This robust and rapid LC/MS/MS assay is now successfully applied for routine therapeutic drug monitoring of CP, thiotepa and their metabolites in our hospital.

Antineoplastic Agents, Alkylating↗
PubMed2004

Simple, sensitive and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric method for the quantification of lacidipine in human plasma.

A simple, sensitive and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric method was developed and validated for the quantification of lacidipine in human plasma using its structural analogue, amlodipine, as internal standard (IS). The method involves a simple single-step liquid-liquid extraction with tert-butyl methyl ether. The analyte was chromatographed on an Xterra MS C(18) reversed-phase chromatographic column by isocratic elution with 20 mM ammonium acetate buffer-acetonitrile (10:90, v/v; pH 6) and analyzed by mass spectrometry in the multiple reaction monitoring mode. The precursor to product ion transitions of m/z 456.4 --> 354.4 and m/z 409.3 --> 238.3 were used to measure the analyte and the I.S., respectively. The chromatographic run time was 1.5 min and the weighted (1/x(2)) calibration curves were linear over the range 0.1-25 ng ml(-1). Lacidipine was sensitive to temperature in addition to light. The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability and re-injection reproducibility. The limit of detection and lower limit of quantification in human plasma were 50 and 100 pg ml(-1), respectively. The within- and between-batch accuracy and precision were found to be well within acceptable limits (<15%). The analyte was stable after three freeze-thaw cycles (deviation <15%). The average absolute recoveries of lacidipine and amlodipine (IS) from spiked plasma samples were 51.1 +/- 1.3 and 50.3 +/- 4.9%, respectively. The assay method described here could be applied to study the pharmacokinetics of lacidipine.

Calcium Channel Blockers↗
PubMed2005

Screening for and validated quantification of phenethylamine-type designer drugs and mescaline in human blood plasma by gas chromatography/mass spectrometry.

In recent years, several newer designer drugs of the so-called 2C series such as 2C-D, 2C-E, 2C-P, 2C-B, 2C-I, 2C-T-2, and 2C-T-7 have entered the illicit drug market as recreational drugs. Some fatal intoxications involving 2C-T-7 have been reported. Only scarce data have been published about analyses of these substances in human blood and/or plasma. This paper describes a method for screening and simultaneous quantification of the above-mentioned compounds and their analog mescaline in human blood plasma. The analytes were analyzed by gas chromatography/mass spectrometry in the selected-ion monitoring mode, after mixed-mode solid-phase extraction (HCX) and derivatization with heptafluorobutyric anhydride. The method was fully validated according to international guidelines. Validation data for 2C-T-2 and 2C-T-7 were unacceptable. For all other analytes, the method was linear from 5 to 500 microg/L and the data for accuracy (bias) and precision (coefficient of variation) were within the acceptance limits of +/-15% and <15%, respectively (within +/-20% and <20% near the limit of quantification of 5 microg/L).

Designer Drugs↗
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↗