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PubMed1990

[Discriminant analysis of quantification theory on prognostic factors of chemotherapeutic effects on advanced testicular cancer. East Japan Testicular Tumor Study Group].

Prognostic factors of chemotherapy against advanced testicular cancer were analysed in 33 cases studied by East Japan Testicular Tumor Study Group. In this study a discriminant analysis of quantification theory (a multivariate analysis) was adapted, taking 7 factors as comparative variables. The significant factors for complete tumor response were clinical stage, histology, and tumor markers (HCG-beta, AFP). Prognostic score was calculated in each case by quantification theory, which correctly discriminated the group with CR from that without CR, at a probability of 90.1%. The results of our study indicate that the outcome of chemotherapy on advanced testicular cancer may be predicted with probability, by patients' status and adopted treatment. It may enable one to make an adequate treatment schedule for each patient.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1996

Quantification of tumor vascularity and flow with amplitude color Doppler sonography in an experimental model: preliminary results.

This study was designed to evaluate a system to quantitate vascularity and tumor blood flow with amplitude (power) color Doppler sonography. The vascularity of nine transplanted murine tumors was determined with quantitated amplitude color Doppler sonography and compared to tumor vascularity estimated by histologic examination. The system used seemed to provide an accurate depiction of the vascularity of tumor vis-àa-vis histologic estimation of vessel density (r = 0.80). Time-activity curves showed greater flow in the experimental group injected with an exotoxin than in the group injected with saline solution. Vascular density quantification with amplitude color Doppler sonography also was more accurate when an intravascular agent (such as an exotoxin) was used than when saline infusions were given. This quantification scheme may allow the development of a system to assess the probability of malignancy and to monitor tumor response to treatment on the basis of the vascularity of the mass.

Adenocarcinoma↗
PubMed1999

Differentiation of small hyperechoic renal cell carcinoma from angiomyolipoma: computer-aided tissue echo quantification.

To assess the value of computer-aided tissue echo quantification in differentiating small hyperechoic renal cell carcinomas from angiomyolipomas, we studied ultrasonographic images of 15 renal cell carcinomas and 20 angiomyolipomas. After digitizing the images, we measured the absolute gray scale values of the renal cortex, central echo complex, and mass. The relative gray scale value (%) of the mass was calculated by setting the gray scale value of the cortex as 0% and the central echo complex as 100%. The relative gray scale value of renal cell carcinomas was in the range of 12 to 73% (mean, 28%), whereas that of angiomyolipomas was 30 to 204% (mean, 130%). The differentiation between small hyperechoic renal cell carcinomas and angiomyolipomas can be facilitated by computer-aided tissue echo quantification.

Adult↗
PubMed1999

Polymorphisms for the size of heterochromatic regions allow sex-independent quantification of post-BMT chimerism targeting metaphase and interphase cells.

BACKGROUND AND OBJECTIVE: Fully quantitative cytological techniques for the analysis of hemopoietic chimerism are very limited and largely restricted to sex-chromosome detection after sex-mismatched bone marrow transplants (BMTs). The aim of the present investigation was to assess the usefulness of autosomal polymorphisms for the size of heterochromatic regions in the identification of donor and recipient cells and therefore in the quantification of the hemopoietic chimerism after sex-matched BMT. DESIGN AND METHODS: Hemopoietic chimerism was followed up in 3 transplanted patients targeting a polymorphism for the size of the pericentromeric heterochromatin (PCH) of chromosome 9, uncovered by restriction endonuclease (RE) in situ digestion (REISD) with the RE Sau3A, to differentiate donor and recipient cells on conventional bone marrow chromosome preparations. RESULTS: The polymorphism for the size of the PCH of chromosome 9 allowed differentiation of donor and recipient cells targeting both metaphase and interphase nuclei. The misidentification error for the polymorphism for the size of HPC of chromosome 9 was estimated as 1% for metaphases and 6-11% for interphases. The 3 cases studied showed complete chimerism in the first post-BMT sample analyzed, which was maintained in 2 of them. One patient relapsed and showed transient mixed chimerism. One month later, this patient achieved a second complete remission, showing complete chimerism again. In this patient, who received a sex-mismatched BMT, chimerism was also quantified by sex-chromosome identification using established methods, such as conventional cytogenetics and FISH, and the results obtained were similar to those rendered by Sau3A-REISD. INTERPRETATION AND CONCLUSIONS: The polymorphism for the size of the PCH of chromosome 9 uncovered by Sau3A-REISD allows accurate quantification of the hemopoietic chimerism after sex-matched BMT.

Bone Marrow Transplantation↗
PubMed1993

Magnetic resonance angiography and blood flow quantification.

A variety of magnetic resonance (MR) techniques are available for flow imaging and quantification, each exploiting a different property of flowing blood to achieve contrast with stationary tissue, and each with its own strengths and limitations dependent on the flow conditions. These same techniques have been used to perform MR angiography of peripheral as well as the coronary arteries. This article provides an overview of MR angiographic and blood flow quantification techniques and their clinical application. Future technical advances in MR in concert with continuing developments in computer software and hardware are likely to make MR a major vascular technique in the coming decade.

Blood Flow Velocity↗
PubMed1999

Iterative reconstruction based on median root prior in quantification of myocardial blood flow and oxygen metabolism.

UNLABELLED: The aim of this study was to compare reproducibility and accuracy of two reconstruction methods in quantification of myocardial blood flow and oxygen metabolism with 15O-labeled tracers and PET. A new iterative Bayesian reconstruction method based on median root prior (MRP) was compared with filtered backprojection (FBP) reconstruction method, which is traditionally used for image reconstruction in PET studies. METHODS: Regional myocardial blood flow (rMBF), oxygen extraction fraction (rOEF) and myocardial metabolic rate of oxygen consumption (rMMRO2) were quantified from images reconstructed in 27 subjects using both MRP and FBP methods. For each subject, regions of interest (ROIs) were drawn on the lateral, anterior and septal regions on four planes. To test reproducibility, the ROI drawing procedure was repeated. By using two sets of ROIs, variability was evaluated from images reconstructed with the MRP and the FBP methods. RESULTS: Correlation coefficients of mean values of rMBF, rOEF and rMMRO2 were significantly higher in the images reconstructed with the MRP reconstruction method compared with the images reconstructed with the FBP method (rMBF: MRP r = 0.896 versus FBP r = 0.737, P < 0.001; rOEF: 0.915 versus 0.855, P < 0.001; rMMRO2: 0.954 versus 0.885, P < 0.001). Coefficient of variation for each parameter was significantly lower in MRP images than in FBP images (rMBF: MRP 23.5% +/- 11.3% versus FBP 30.1% +/- 14.7%, P < 0.001; rOEF: 21.0% +/- 11.1% versus 32.1% +/- 19.8%, P < 0.001; rMMRO2: 23.1% +/- 13.2% versus 30.3% +/- 19.1%, P < 0.001). CONCLUSION: The MRP reconstruction method provides higher reproducibility and lower variability in the quantitative myocardial parameters when compared with the FBP method. This study shows that the new MRP reconstruction method improves accuracy and stability of clinical quantification of myocardial blood flow and oxygen metabolism with 15O and PET.

Adult↗
PubMed1999

[Ultrasonography contrast media Levovist and power Doppler in the study of the breast. Methodology, vascular morphology and automatic enhancement quantification with wash-in and wash-out curves].

PURPOSE: To define the technique for contrast-enhanced power Doppler US studies of breast lesions and to identify possible clinical applications. MATERIAL AND METHODS: We studied 51 breast lesions detected at mammography and confirmed at cytology and/or surgical biopsy; 15 were benign lesions and 36 carcinomas, namely 14 T1ab, 29 T1c and 8 T2. We found 14 masses with regular margins, 28 with irregular margins, 1 asymmetric density, 2 architectural distortions and 6 clustered calcifications. US studies were performed with an AU5 Harmonic unit (Esaote Biomedica, Genoa, Italy) equipped with a software for online image storage, analysis and automatic quantification of US signal intensity changes after contrast agent injection, namely wash-in and wash-out contrast enhancement curves. The echocontrast agent Levovist (Schering AG, Berlin, Germany), 4.0 g preparation, was administered by i.v. injection (cubital vein) in two times at a concentration of 400 mg/mL. The first 4 mL of Levovist suspension were injected as a bolus at approximately .5 mL/s to evaluate lesion vascularization and choose the best scanning plane for wash-in and wash-out quantification. The remaining 6 mL of Levovist suspension were injected at approximately 1.0 mL/s and dedicated to wash-in and wash-out recording. RESULTS: The region of interest could not be identified in 2 of 6 calcifications. After Levovist administration, signal enhancement was seen in 36 lesions. Nonsignificant curves were obtained in 7 fibroadenomas, 1 fibrocystic mastopathy and 5 carcinomas. Pathology diagnosed an in situ component around the lesion core (true positives) in 12 carcinomas with perilesional vessels and also 3 more carcinomas with perilesional foci in situ missed at contrast-enhanced US (false negatives). The wash-in/wash-out curves of 30 carcinomas differed from those of the 6 fibroadenomas, in that the former had faster wash-in and an earlier enhancement peak, as well as longer enhancement than the latter. Moreover, fibroadenoma curves are regularly increasing, with moderate variations. As for wash-out, carcinomas exhibited three main patterns, namely a monophasic, a polyphasic and a plateau pattern. DISCUSSION: The pattern of enhancement curves in fibroadenomas is related to straight and regular vessels, while arteriovenous shunts in carcinomas cause early signal intensity peaks. Wash-out is longer in carcinomas than in fibroadenomas because the former present anarchic and tortuous vessels with slow flows. CONCLUSIONS: Levovist enhanced US is a complementary test to study known breast lesions which permits the differential diagnosis of carcinomas and fibroadenomas. Our results justify a larger clinical trial to assess the role of this technique for diagnosis, prognosis and staging purposes.

Adult↗
PubMed1999

DNA quantification as a prognostic factor in colorectal adenocarcinoma.

OBJECTIVE: To determine if DNA quantification, studied in cytologic samples obtained by fiberendoscopy, has predictive value in colorectal adenocarcinoma. STUDY DESIGN: Thirty patients with colorectal cancer diagnosed by cytology, endoscopy and microbiopsy, were selected. The study covered > 10 years. Smears were stained with progressive hematoxylin and processed by computer for DNA evaluation by image cytometry. RESULTS: Four different types of histograms that directly related to malignancy were obtained. These histograms were characterized by the value of entropy, and we established four grades of aggressiveness. Finally, we obtained two large groups: high and low grade of malignancy. We studied the survival times in both groups and used a Kaplan-Meier survival curve for the high grade group. We confirmed the results statistically and found that there was a significant relationship, with P < .05. CONCLUSION: We strongly advise the use of DNA quantification by image cytometry in daily surgery as a prognostic indicator of survival time in colorectal cancer.

Adenocarcinoma↗
PubMed2000

Cardiac output quantification by Doppler echocardiography in intensive care--limitations and validation.

UNLABELLED: Cardiac output (CO) quantification is primordial to the evaluation of patients with heart failure who are on tailored therapy and under invasive hemodynamic monitoring. Doppler echocardiography can be used to access CO noninvasively, but the concordance between its results and those obtained by invasive methods in paired measurements is still controversial. To our knowledge, no previous studies have assessed the clinical relevance of Doppler echocardiography for CO serial evaluation in patients submitted to tailored therapy. AIM: To evaluate the usefulness of echo-Doppler in the assessment of CO and quantification of changes in CO, compared to thermodilution, in patients with advanced heart failure under hemodynamic monitoring to guide tailored therapy. METHODS: In 20 patients (14 male, 62 +/- 14 years old, all in sinus rhythm), with dilated cardiomyopathy and NYHA IV, admitted to the intensive care unit (ICU), CO was simultaneously determined by Doppler echocardiography (dpCO) and thermodilution (tdCO) in three serial evaluations (overall 60). The dpCO was calculated by multiplying the aortic orifice area by the velocity-time integral of aortic continuous wave Doppler flow and by the heart rate. A difference between tdCO and dpCO of more than 20% was considered a major error. RESULTS: In the overall evaluations, dpCO systematically overestimates tdCO (p = 0.026). The correlation between tdCO and dpCO was 0.81, the mean difference between measurements was 0.40 +/- 0.61 l/min (mean -2SD = -1.62 mean +2SD = 0.81) and 19 (32%) major errors occurred. No significant difference was found between CO percentual variation values assessed by both methods, with a stronger correlation (r = 0.92-p = 0.014) compared to that found for absolute values. On using the dpCO/tdCO ratio in the first evaluation to correct subsequent dpCO, the correlation was fairly good (r = 0.96-p = 0.0002 versus corrected dpCO). The mean difference between paired measurements was significantly lower (0.12 +/- 0.28 l/min-mean 2SD = -0.44 mean +2SD = 0.67), and there were no errors. CONCLUSIONS: CO estimated by Doppler echocardiography has a good correlation with thermodilution although with a weak concordance between paired results in patients with dilated cardiomyopathy and advanced heart failure admitted to the ICU for tailored therapy. Our results with dpCO percentual change in repeated evaluations and with corrected dpCO value after a single simultaneous invasive determination suggest that Doppler echocardiography is a valid method for clinical purposes, allowing us to propose a reduction in the time period of invasive hemodynamic monitoring.

Adult↗
PubMed2000

Improved quantification of cell survival on stromal monolayers by flow cytometric analyses.

BACKGROUND: The ex vivo survival of leukemic cells maintained on bone marrow stroma is an important tool for the investigation of cell survival and leukemogenesis. Currently, ex vivo survival of leukemic cell survival is measured by coculture on stromal cell monolayers. In these assays, we postulated that two important sources of error might be introduced through either variations in flow volume or in donor stromal cells. METHODS: A previously reported coculture assay that maintains leukemic cells on bone marrow stromal cells was employed. RESULTS: We identified two means of optimizing the coculture assay. First, biologically inert beads having well-characterized fluorescent properties were added to each sample to mathematically adjust for flow-based variations in volume acquisition. The inclusion of fluorescent beads to the basic stromal cell assay showed a significantly lower coefficient of variation as compared to samples analyzed without beads or manually counted using a hemacytometer. Second, in order to minimize variability in bone marrow hematopoietic function between donors, an adherent stromal cell line known to support hematopoiesis (HS-5) was used. When normal human donor stromal cells were used, variability in the survival of leukemic cells was observed on stromal cells derived from different donors. In contrast, statistically significant variability in survival of leukemic cells was not seen on HS-5 monolayers. Finally, we demonstrate that patient-derived leukemic samples may be examined for cell survival using these modifications. CONCLUSIONS: The novel use of fluorescent beads and a hematopoietic-supportive stromal cell line together makes the quantification of stroma-supported cell survival more reproducible, accurate, and amenable to patient-derived samples. These improvements in flow cytometry-based cell quantification are an important step in establishing a role for stromal cell assays in the study of leukemia biology and therapy.

Bone Marrow Cells↗
PubMed2000

Simultaneous quantification of various retinoids by high performance liquid chromatography: its relevance to alcohol research.

BACKGROUND: We established a high performance liquid chromatography system that allowed simultaneous quantification of various retinoids. METHODS: We applied the retinoids to a high performance liquid chromatography system with a silica gel absorption column. Samples were separated by the system with a binary multistep gradient with two kinds of solvent that contained n-Hexan, 2-propanol, and glacial acetic acid in different ratios. Each retinoid was detected at a wavelength of 350 nm. RESULTS: This condition allowed separation of 13-cis-retinoic acid, 9-cis-retinoic acid, all-trans-retinoic acid, 13-cis-retinol, all-trans-retinol, all-trans-4-oxo-retinoic acid, and 13-cis-4-oxo-retinoic acid as distinct single peaks. Each retinoid was also analyzed separately and its retention time determined. To ascertain the reliability of this system for retinoid quantification, retinoids at various concentrations were applied to the system. We observed the linearities between the concentration and area under the curve of the peak for each retinoid by linear least-squares regression analysis up to 2.5 ng/ml for all retinoic acids and up to 5 ng/ml for all retinols. There was no significant scattering in tests of within-day reproducibility or day-to-day reproducibility. Using this system, we examined effects of light exposure on isomerization of retinoids. When retinoids were exposed to room light for 2 hr, the amounts of all but 13-cis-retinol changed significantly. In particular, the amounts of all-trans-retinoic acid and 9-cis-retinoic acid were reduced by 40% and 60%, respectively. CONCLUSION: The HPLC system established in this study should be useful for studying the oxidation pathway of retinol to retinoic acid. A light-shielded condition is required when particular retinoic acids are analyzed.

Alitretinoin↗
PubMed2000

[Assessment of most appropriate background subtraction method for quantification of 123I-metaiodobenzylguanidine (MIBG) myocardial uptake by comparing with plasma ANP and BNP].

BACKGROUND: Quantification of 123I-metaiodobenzylguanidine (MIBG) myocardial uptake is widely accepted as a useful tool for estimating the severity of congestive heart failure. However, most reliable method has not been determined yet because of the difficulty of background (BG) subtraction. In this study, the most appropriate BG subtraction method was evaluated as compared with plasma atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), norepinephrine (NEP) and left ventricular ejection fraction (LVEF). METHODS: Twenty-one patients with chronic heart failure were examined. After bolus injection of 123I-MIBG (111 MBq), dynamic anterior chest images were collected every second for 2 minutes. Planar anterior chest images were obtained 15 minutes (early image) and 4 hours later (delayed image) respectively. The parameters for quantification of 123I-MIBG myocardial uptake used in this study were heart to mediastinum ratio (H/M), myocardial washout rate and uptake ratio. Background was calculated using the region of interest (ROI) placed over a part of upper mediastinum, whole upper mediastinum, right lung and pericardial space respectively. The values of these parameters were calculated with and without BG subtraction and compared with plasma ANP, BNP and others. RESULTS: H/M did not correlate with ANP or BNP. Myocardial washout rate without BG subtraction showed significant correlation with ANP (p < 0.01) and BNP (p < 0.05). After BG subtraction employing ROI placed over the part of upper mediastinum and whole upper mediastinum, myocardial washout rate showed better correlation with BNP (p < 0.01). Myocardial uptake ratio did not showed any correlation with ANP or BNP without BG subtraction. However, myocardial uptake ratio showed significant correlation with BNP (p < 0.05) after subtraction of upper mediastinal BG. BG subtraction using ROI over right lung or pericardial area revealed poor results in both myocardial washout rate- and uptake ratio. CONCLUSION: BG subtraction using ROI over the upper mediastinum is likely to be suitable for quantitative analysis of 123I-MIBG myocardial scintigram.

3-Iodobenzylguanidine↗
PubMed2000

Development and validation of a method for simultaneous separation and quantification of 5 different sugars in canine urine.

The objective of this project was to develop and validate a method for concurrent separation and quantification of methylglucose, rhamnose, xylose, sucrose, and lactulose in canine urine by using high pressure anion exchange liquid chromatography and pulsed amperometric detection. The method was validated by evaluating dilutional parallelism, spiking recovery, intra-assay variability, and inter-assay variability. Observed to expected ratios for 3 urine samples, and all sugars, ranged from 77.6% to 106.9% for a 1:2 dilution, 85.2% to 121.4% for a 1:4 dilution, and 91.6% to 163.7% for a 1:8 dilution. Observed to expected ratios for spiking recovery of 3 urine samples, all sugars, and 5 different spiking solutions, ranged from 85.5% to 116.7 % (mean +/- SD, 100.5 +/- 6.0%). The intra-assay coefficients of variation were 1.6%, 3.4%, and 4.7% for methylglucose; 1.6%, 2.0%, and 3.6% for rhamnose; 2.7%, 1.4%, and 1.1% for xylose; 9.8%, 3.4%, and 4.0% for sucrose; and 3.2%, 3.3%, and 3.3% for lactulose. Inter-assay coefficients of variation were 3.2%, 5.7%, and 4.2% for methylglucose; 4.3%, 5.4%, and 6.4% for rhamnose; 3.3%, 5.0%, and 4.2% for xylose; 9.4%, 9.9%, and 9.4% for sucrose; and 6.1%, 4.9%, and 2.7% for lactulose. In conclusion, a method for simultaneous separation and quantification of 5 sugars in canine urine was established and found to be linear, accurate, precise, and reproducible. This method may prove useful in the simultaneous evaluation of gastric permeability, small intestinal permeability, and small intestinal mucosal function in dogs with gastrointestinal disorders.

Animals↗
PubMed2000

[Quantification of iron overload].

Iron overload involves primarily hepatocytes in case of digestive hyperabsorption (hemochromatosis and dyserythropoiesis) and macrophages in case of transfusional excess. Serum iron and transferrin saturation are poorly correlated with the degree of iron overload. Serum ferritin is a better reflect of iron stores but numerous clinical conditions, unrelated to variations of iron load, can increase the serum level. Biochemical determination of liver iron overload is the gold standard of iron quantification and well correlated to the level of iron burden appreciated by the amount of iron removed by venesection, but its determination necessitates a liver biopsy and is dependant of sampling error in case of heterogeneous iron deposits (cirrhosis). The sensitivity of computed tomography is insufficient, beeing unable to detect iron overload below 5 times the normal liver iron load, especially in case of associated steatosis. Magnetic resonance imaging is a valuable tool when using T2 weighted gradient echo sequences on 1.5 Tesla magnet and permits non invasive iron overload quantification.

Biopsy↗
PubMed2000

Quantification and reproducibility of tracking cortical extent of activation by use of functional MR imaging and magnetoencephalography.

BACKGROUND AND PURPOSE: Functional MR imaging and magnetoencephalography are commonly used to study normal cortical sensory and cognitive processing as well as a variety of disease states. The usefulness of these techniques is dependent on the reproducibility and sensitivity to change of derived measures of brain function. The purpose of this study was to compare the efficacy of functional MR imaging and magnetoencephalography as measures of the extent of cortical activity in response to a graded stimulus. METHODS: Five participants underwent functional MR imaging and magnetoencephalography involving stimulation of one, two, three, and four digits of the left hand. Measurements of activation were repeated three times per participant. The cortical extent of activation was assessed for functional MR imaging by observing the number of "activated" pixels and the "amount of activation": the product of the number of activated pixels and the mean signal change. Activation was quantified for magnetoencephalography as the magnitude of the evoked magnetic field peak and as the strength of the modeled current source, Q. RESULTS: For functional MR imaging, the number of activated pixels tended to increase with the increasing number of stimulated digits. High intra- and interparticipant variability (66% and 85% variation, respectively) did not, however, allow statistical resolution of this trend. The amount of activation was similarly variable (interparticipant, 89%). Magnetoencephalography was more robust regarding quantification. The evoked field amplitude varied linearly with the number of digits stimulated; intra- and interparticipant variability was 18% and 41%, respectively, permitting resolution of significant differences between any combination of stimulated digits, except two versus three (P < .05). CONCLUSION: Although functional MR imaging and magnetoencephalography show measurable evoked responses with somatosensory stimulation, in this study, functional MR imaging did not permit robust quantification of increasing cortical areas of activation.

Cerebral Cortex↗
PubMed2000

Influence of DNA target melting behavior on real-time PCR quantification.

BACKGROUND: Quantitative real-time PCR is increasingly used to quantify copy numbers of nucleic acids for clinical applications. We observed that the measurements of allele imbalances of the tumor suppressor gene p16 and the oncogene ErbB-2 yielded results with variable precision under certain experimental conditions. METHODS: We used the LightCycler(TM) real-time PCR system to quantify different genomic target sequences using hybridization probes or SYBR Green for detection. RESULTS: With two primer/template systems (p16 and ErbB-2), we observed sinusoidal scattering of the threshold cycle values depending on the capillary position in the thermostated reaction chamber. This scattering depended on the denaturation temperature only when complete genomic DNA was used as template and did not occur when PCR product or restricted or boiled genomic DNA was used or the denaturation temperature in the first cycles was increased (and other targets, such as p53, HBB, IGF-1, GAPDH, and PBGD, did not show this behavior). CONCLUSIONS: Before a primer system is used for precise quantitative real-time PCR, the dependence of the quantification results on the positions of reaction tubes in the thermocycler should be tested. Our data indicate that amplification efficiencies, especially in the first cycles, depend not only on the priming efficiencies of the primers and the melting temperature of the amplicon, but also on the melting behavior of the amplicon's genomic vicinity. Complete denaturation of genomic DNA is necessary to maximize precision of quantitative PCR. Higher denaturation temperatures in the initial cycles or boiling of DNA before the PCR can improve the accuracy of quantification in some cases.

Cyclin-Dependent Kinase Inhibitor p16↗
PubMed2001

Simultaneous absolute quantification of target and control templates by real-time fluorescence reverse transcription-PCR using 4-(4'-dimethylaminophenylazo)benzoic acid as a dark quencher dye.

BACKGROUND: Despite the many advantages of real-time fluorescence reverse transcription-PCR (RT-PCR) as a quantitative analytical tool, simultaneous quantification of target and reference templates within one reaction has not been reported. We developed such an assay with an internal reference template. METHODS: For quantification of target and reference sequences, we used two fluorescent probes in one reaction vessel on an ABI PRISM 7700 SDS instrument. Fluorescent probes were labeled with either 6-carboxy-fluorescein or hexachloro-6-carboxy-fluorescein as reporter dye and 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL) as a dark quencher fluorophore. To test the sensitivity and specificity of this assay, serial dilutions of reference and target templates were analyzed in one PCR reaction. In the presence of 10 beta-actin molecules as control templates, 10(5) bcr/abl molecules were amplified, and 10(5) beta-actin molecules were amplified in the presence of 10 bcr/abl copies. We also performed single and duplex measurements on samples from five patients with documented Philadelphia chromosome-positive chronic myelogenous leukemia disease courses (72 samples) and three with minor bcr/abl+ acute myelogenous leukemias (26 samples). RESULTS: For M-bcr/abl duplex RT-PCR, the correlation coefficient (r) for starting template amounts and threshold cycle values was 0.99; for m-bcr/abl, r = 0.96, indicating a precise log-linear relation for 10-10(5) copies/100 ng of cDNA. In the same PCR reactions, r = 0.99 for beta-actin (coamplified with M-bcr/abl or m-bcr/abl) for 10(3)-10(7) copies/100 ng cDNA. The linear correlation coefficient for single and duplex measurements was 0.98 for M- and m-bcr/abl in patient samples. CONCLUSIONS: DABCYL can be used as dark quencher fluorophore in real-time fluorescence PCR. The duplex fluorescence RT-PCR assay for bcr/abl and beta-actin transcripts allows monitoring of bcr/abl+ leukemias.

Fluorescent Dyes↗
PubMed2001

MALDI-TOF MS for quantification of substrates and products in cultivations of Corynebacterium glutamicum.

The application of MALDI-TOF MS for the quantification of lysine, alanine, and glucose is described. The method is based on using stable isotopes as internal standards and allows fast, sensitive, and reproducible quantification of these compounds. It is demonstrated for aqueous standard solutions with concentrations of the analytes between 10 microM and 100 mM. The mean standard deviations from five replicates each were 4.3% (lysine), 3.7% (alanine), and 3.2% (glucose). In addition, sucrose could be measured by MALDI-TOF MS, but was not quantified due to lack of an internal standard. The method developed can be applied to quantify metabolites in cultivations of C. glutamicum ATCC 21253, without processing of the sample except a 1:5 dilution. Excellent agreement of the data with conventional techniques like HPLC or enzyme assay was found.

Cell Culture Techniques↗