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PubMed2001

[Effects of proteolytic enzymes and sexual abstinence on alpha glucosidase biochemical quantification in human seminal plasma].

BACKGROUND: alpha-glucosidase is found in human seminal plasma as an acid form, located in accessory glands, and as a neutral form secreted almost exclusively by the epididymis. Quantification of alpha-glucosidase activity is a marker of the secretory function of the epididymis and indemnity of the sperm transport pathway. AIM: To obtain reference values for alpha-glucosidase in normal samples of seminal plasma, to evaluate its behavior in serial samples and to determine the effect of proteolytic enzymes. MATERIAL AND METHODS: Fifty donors, with normal semen analysis according to the criteria of the World Health Organization, were evaluated. For the study with alpha-quimotrypsin, 0.1 to 10 mg/ml of the enzyme was added to the seminal plasma from a group of donors. alpha-glucosidase was also measured in semen obtained from nine patients at different time intervals. RESULTS: Normal alpha-glucosidase values ranged from 14.52 to 25.69 microU/ml. Concentrations up to 10 mg/ml of alpha-quimotrypsin (10 times of that usually used in the liquefaction of the semen) did not alter the quantification of alpha-glucosidase. Serial determinations revealed oscillations in their magnitude, which stayed in each patient's characteristic range. However a subgroup presented a marked reduction of the activity of alpha-glucosidase as the abstinence diminished (40%). CONCLUSIONS: Evaluation of alpha-glucosidase in seminal plasma gives reliable information of the secretor state of the epididymis and especially replaces invasive methods used to evaluate the indemnity of the spermatic transport from the epididymis to the anterior urethra.

Adult↗
PubMed2001

Sensitive and rapid quantification of busulfan in small plasma volumes by liquid chromatography-electrospray mass spectrometry.

BACKGROUND: High-dose busulfan is widely used in conditioning regimens before hematopoietic stem cell transplantation in both adults and children. Large interindividual variability in pharmacokinetics after oral administration has been reported; therefore, therapeutic drug monitoring of busulfan may decrease the incidence of drug-related toxicity (for example, hepatic venoocclusive disease) and may also improve therapeutic efficacy. METHODS: Busulfan concentrations were quantified using 200 microL of plasma and liquid-liquid extraction with diethyl ether after the addition of [2H8]busulfan as the internal standard. Separation and detection of busulfan and [2H8]busulfan were achieved with a LUNA C8 column (5 microm; 150 x 2 mm i.d.) at 30 degrees C, a HP 1100 liquid chromatography system, and a HP 1100 single-quadrupole mass spectrometer. Busulfan and [2H8]busulfan were detected as ammonium adducts in selected-ion monitoring mode at m/z 264.2 and 272.2, respectively. RESULTS: The calibration curve was linear at 5-2000 microg/L busulfan. Intra- and interassay imprecision (CV) and bias were both <11%. The limits of detection and quantification were 2 and 5 microg/L, respectively. Extraction recovery of busulfan was >87%. Analysis of pharmacokinetics in four patients receiving high-dose busulfan indicated that minimum busulfan concentrations before the next dose were 405-603 microg/L, with no interference observed. CONCLUSIONS: The new rapid and sensitive liquid chromatographic-mass spectrometric assay is an appropriate method for quantification of busulfan in human plasma, making therapeutic drug monitoring of busulfan faster and easier in clinical practice.

Adolescent↗
PubMed2001

Quantification of serotonin transporters in nonhuman primates using [(123)I]ADAM and SPECT.

UNLABELLED: We reported recently a highly selective radioligand, 2-([2-([dimethylamino]methyl)phenyl]thio)-5-[(123)I]iodophenylamine (ADAM), for SPECT imaging of serotonin transporters (SERT). In this article we describe the kinetic modeling of [(123)I]ADAM and its ability to quantitatively and reproducibly measure the concentrations of SERT in the nonhuman primate brain. We also investigate simplified models of tracer behavior that do not require invasive arterial blood sampling. METHODS: Three female baboons each underwent 3 [(123)I]ADAM SPECT studies. The studies consisted of a dynamic sequence of seventy-two 5-min scans after injection of 330 +/- 50 MBq (mean +/- SD) [(123)I]ADAM. Rapid arterial blood samples were obtained and corrected for the presence of labeled metabolites. Dynamic imaging and metabolite-corrected plasma data were analyzed using graphic analysis to give the distribution volumes (DVs) of different brain regions. DV ratios (DVRs) of target to cerebellum were derived and compared against a kinetic reference tissue model and simple target-to-background ratio. RESULTS: Averaged over all 9 scans, the mean DV in the midbrain was 4.86 +/- 1.06 mL/mL and the mean DV in the cerebellum was 2.25 +/- 0.48 mL/mL. The mean test-retest repeatability of the midbrain DV was 14.5%. The reference tissue model gave a mean midbrain DVR of 2.01 +/- 0.17 and correlated strongly with the DVR calculated from the full kinetic model (correlation coefficient [R(2)] = 0.94; P < 0.001), but with much improved repeatability (test-retest, 5.4%; intersubject variability, 5.2%). Similarly, the simple ratio method gave strong correlations with the full kinetic model (R(2) = 0.89; P < 0.001) and a test-retest of 7.6%. CONCLUSION: Accurate, repeatable quantification of SERT in the nonhuman primate brain is possible using kinetic modeling of dynamic [(123)I]ADAM SPECT scans. Simplified models, which do not require arterial blood sampling, gave accurate results that correlated strongly with the full kinetic model. The test-retest reliability of the simplified reference region models was excellent. Quantification of SERT is possible using full kinetic modeling and also with simpler reference region methods.

Animals↗
PubMed2001

[A research on the quantification of retinal blood vessels in cases of arteriovenous crossing pressure syndrome caused by hypertension].

The method for quantification of retinal blood vessels in cases of arteriovenous crossing pressure syndrome caused by hypertension was studied. The matched filtering pre-processing of image enhancement for local retinal blood vessels and closing of mathematical morphology for image smoothing, and the method of scale-up lineation for contour determination of retinal blood vessels and optimal seeking algorithm with reversible quantification were used to measure the pathological deformation of vessel, thus providing a computer method for auxiliary diagnosis of relative diseases.

Algorithms↗
PubMedDate not supplied

[Quantification of T CD4+ lymphocytes and viral RNA in patients with HIV/AIDS].

Quantification of CD4+ T lymphocytes and viral RNA in patients with HIV/AIDS. Levels of progression markers (viral load and CD4+ T lymphocytes) in 410 patients with HIV/AIDS that were in different clinical stages of the disease and under different therapeutic schemes were quantified. The objective was to determine the correlation between values of progression markers and clinical stage of the patients. Commercial methodologies for the quantification of lymphocytes, subpopulations and circulating viral RNA were used. Results indicate that there was a correlation between low CD4+ values and high viral load in patients with antiretroviral treatment but not in patients without treatment. Furthermore, analysis of 1,208 samples processed during 1999 showed that 46% of the patients had less than 200 CD4+ T lymphocytes/mL blood and more than 500 copies of circulating viral RNA. Implications of these results in public health in Mexico are discussed.

CD4 Lymphocyte Count↗
PubMed2002

Simultaneous quantification of prostate-specific antigen and human glandular kallikrein 2 mRNA in blood samples from patients with prostate cancer and benign disease.

BACKGROUND: Detection or quantification of circulating cancer cells has been proposed as an aid in detection and monitoring of several solid tumors. We investigated the classification accuracy of prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) mRNA copy numbers in blood for the differentiation of patients with prostate cancer (PC) and benign disease. METHODS: PSA and hK2 mRNA expression was studied in blood samples from 51 men with PC and 19 men with benign disease. Among the PC patients, 10 had organ-confined disease (pT1-pT2). We used a multiplexed reverse transcription-PCR assay with two highly target-like mRNA internal standards for the simultaneous quantification of PSA and hK2 mRNA. An external calibration curve covered the range of 10(2)-10(6) mRNA copies. RESULTS: PSA and hK2 mRNA were detected in 41 of 51 (median, 1200 copies/0.5 mL of blood) and 43 of 51 (median, 3800 copies/0.5 mL of blood) patients with PC, respectively, whereas only 1 of 19 men with benign disease was positive for both mRNAs (1500 PSA and 3100 hK2 mRNA copies/0.5 mL of blood; P <0.0001, Mann-Whitney U-test). Of the 10 patients with organ-confined PC, only 3 with low Gleason scores (< or =5) were negative for both PSA and hK2 (P = 0.02, Mann-Whitney U-test). CONCLUSIONS: The presence of PC cells in the blood circulation is an early event in PC progression, and quantitative assays for PSA and hK2 mRNA discriminate benign from PC cases. Further studies are needed to determine the diagnostic accuracy and prognostic value of the assays.

Aged↗
PubMed2002

Absolute quantification of myocardial blood flow with H(2)(15)O and 3-dimensional PET: an experimental validation.

UNLABELLED: The purpose of this study was to assess a 3-dimensional (3D)-only PET scanner (ECAT EXACT3D) for its use in the absolute quantification of myocardial blood flow (MBF) using H(2)(15)O. METHODS: Nine large white pigs were scanned with H(2)(15)O and C(15)O before and after partially occluding the circumflex (n = 4) or the left anterior descending (n = 5) coronary artery at rest and during hyperemia induced by intravenous dipyridamole. Radioactive microspheres labeled with either (57)Co or (46)Sc were injected during each of the H(2)(15)O scans, which allowed comparison between microsphere and PET measurements of regional MBF. PET analyses of 3D acquisition data were performed using filtered backprojection reconstruction and region-of-interest definition by factor and cluster analysis techniques and single-compartment model quantification. RESULTS: The Hanning filter applied in image reconstruction resulted in a left atrial blood volume recovery factor of 0.84 +/- 0.06. Differences between repeated measurements of recovery were small (mean, -0.8%; range, -6.6% to 3.6%). In 256 paired measurements of MBF ranging from 0.05 to 4.4 mL. g(- 1). min(-1), microsphere and PET measurements were fairly well correlated. The mean difference between the 2 methods was - 0.11 mL. g(-1). min(-1) and the limits of agreement (+2 SD) were -0.82 and 0.60 mL. g(-1). min(-1) (Bland-Altman plot). CONCLUSION: Dynamic measurements with H(2)(15)O using a 3D-only PET tomograph provide reliable and accurate measurements of absolute regional MBF over a wide flow range. The 3D acquisition technique can reduce the radiation dose to the subject while maintaining adequate counting statistics.

Animals↗
PubMed2002

Flow cytometric quantification of competitive reverse transcription-PCR products.

BACKGROUND: Competitive PCR of reverse transcribed mRNA sequences is used to quantify transcripts, but the usual approaches are labor-intensive and time-consuming. We describe the non-gel-based quantification of competitive reverse transcription (RT)-PCR products with use of microparticles and flow cytometry. METHODS: PCR products of a target sequence and an internal control sequence (competitor) were labeled during PCR using digoxigenin (DIG)- and dinitrophenol (DNP)-labeled primer, respectively, allowing specific binding to microparticles coated with the corresponding antibody. Both amplification products were biotinylated to enable fluorescence labeling with streptavidin-R-phycoerythrin. The mean fluorescence intensity of each microparticle population, corresponding to the amount of bound PCR product, was measured in a flow cytometer. We constructed microparticles coated with antibodies against DIG and DNP to specifically capture PCR products derived from target and competitor sequences, respectively. RESULTS: As required for a reliable competitive PCR assay, nearly identical kinetics were found for the amplification of target and competitor sequences when using only one competitive primer. The method was applied to examine interleukin-8 expression in human lymphocytes after x-irradiation. One hour after irradiation, the concentration of transcripts decreased by half. CONCLUSIONS: The flow cytometric assay for the quantification of competitive RT-PCR products avoids additional hybridization steps and antibody labeling. The use of paramagnetic microparticles would also enable the complete automation of this method.

Digoxigenin↗
PubMed2002

Simple method for quantification of Bence Jones proteins.

BACKGROUND: Quantification of free monoclonal light chains in urine [Bence Jones proteins (BJPs)] is used to diagnose multiple myeloma and to evaluate response to treatment. We have developed and evaluated an optimized approach for quantification of BJPs. METHODS: High-resolution gel electrophoresis of unconcentrated urine and albumin calibrators was carried out on Sebia's Hydrasys instrument with Hydragel HR agarose gels. After staining with acid violet, the gels were scanned densitometrically. The staining intensities of BJP bands relative to the staining intensities of albumin solutions were used to determine the BJP concentrations. Results for patient samples were compared with conventional agarose gel electrophoresis on concentrated samples. RESULTS: The relationships between staining intensity and the protein concentrations of albumin and BJPs were linear up to protein concentrations of approximately 2000 mg/L. The detection limit was approximately 20 mg/L. The interassay imprecision (CV) was approximately 8% (n = 23, duplicate analysis), and the results (y) showed a close positive relationship to the comparison method: slope = 0.82 (confidence interval, 0.75-0.88); y-intercept = 34 (-14 to 81) mg/L; n = 29; r(2) = 0.96. CONCLUSIONS: Agarose gel electrophoresis of unconcentrated urine samples together with a series of albumin calibrators followed by acid violet staining and densitometric scanning is sufficiently reproducible and sensitive to quantify clinically relevant BJPs.

Bence Jones Protein↗
PubMed2003

Automated biofilm morphology quantification from confocal laser scanning microscopy imaging.

In spite of the immediate visual appeal of confocal laser scanning microscopy images, the extraction of accurate reconstitutions of biofilm morphology requires a lengthy and computational intensive succession of processing steps. However, once performed, it provides ample reward by enabling the quantitative study of biofilm structure. A software suite of image processing tools for full automation of biofilm morphology quantification was developed by integrating preprocessing, segmentation and morphology quantification operations. This software toolbox was implemented in a web server and a user friendly interface was developed to facilitate image submission, storage and sharing, its access being unrestricted for scientific applications. The image bioinformatics tool which results from the integration of the processing operations can be accessed at http://www.itqb.unl.pt:111/clsmip/. Its use is described in this paper and is illustrated with an example of processing of experimental data describing the growth of a mixed species dentrifying biofilm.

Automation↗
PubMed2003

[Quantification of urinary hemoglobin by an immunological method].

The quantification method of urinary hemoglobin has not been established. We examined whether a reagent (Eiken, Tokyo) used to immunologically assay fecal hemoglobin could be utilized to quantify urinary hemoglobin. The coefficients of variation were 2.2-3.0% in the reproducibility of one-run assays using urine and 4.3-5.7% in that of multiple-run assays using the standard sample of hemoglobin. Urinary hemoglobin was unstable and decreased in a time- and temperature-dependent manner. However, addition of the hemoglobin stabilization buffer (50 mM phosphate buffer, pH 6.4) (Eiken) to urine made urinary hemoglobin stable. Urinary hemoglobin levels did not change significantly when stored at 4 degrees C for 7 days or at -80 degrees C for 30 days. The hemoglobin concentration (mean +/- SD) of urine showing 1+, 2+ and 3+ with a test strip was 385 +/- 165(n = 30), 1070 +/- 499(n = 40) and 4130 +/- 2770 ng/ml (n = 20), respectively. Urinary hemoglobin did not correlate with urinary albumin, transferrin, immunoglobulin G, N-acetyl-beta-D-glucosaminidase nor alpha 1 microglobulin. These results suggest that this immunological method using the hemoglobin stabilization buffer can be utilized for quantification of urinary hemoglobin, which may provide clinically important information.

Biomarkers↗
PubMedDate not supplied

Real-time PCR (TaqMan PCR) quantification of carcinoembryonic antigen (CEA) mRNA in the peripheral blood of colorectal cancer patients.

Reverse transcriptase-polymerase chain reaction (RT-PCR) has been utilized to detect living micrometastases of cancer cells in the lymph node, ascites or circulation system. However, the method was so sensitive that false-positives happened frequently. Therefore we have developed a quantification of CEA mRNA using real-time PCR to detect living cancer cells in the circulating blood and examined its usefulness as a predictive marker for liver metastases of colon cancer. In cell spiking experiments, it was possible to detect CEA mRNA in 10(1) cancer cells diluted in 10(7) normal lymphocytes. In the blood samples of cancer patients, the CEA mRNA level was significantly higher in Dukes' D patients than in the other clinical stages of colorectal cancer. This indicates that quantification of CEA mRNA is useful for the evaluation of colorectal cancer progress and that the post-operative increase of CEA mRNA can be a predictive marker for micrometastasis.

Adenocarcinoma↗
PubMed2003

Quantification of hippocampal signal intensity in patients with mesial temporal lobe epilepsy.

BACKGROUND: Refractory mesial temporal lobe epilepsy (MTLE) is frequently associated with hippocampal atrophy (HA) and an abnormal hippocampal signal (Hsig) on magnetic resonance imaging (MRI). OBJECTIVE: To quantify Hsig on MRI using a simplified technique. METHODS: The authors included 15 patients with refractory MTLE who underwent surgery and had preoperative MRI with hippocampal volumetry. Hsig was quantified on preoperative coronal T1-weighted and T2-weighted MRI using the NIH-Image program. Hsig was determined for the head, tail, and entire hippocampal extension. Abnormal Hsig was defined when values were above (for T2) or below (for T1) 2 standard deviations from the mean of normal control group. RESULTS: The lateralization of abnormal Hsig values was concordant with electroencephalograms and HA in all patients. There was a significant difference in ipsilateral T2 Hsig between patients and controls (P < .0001), but not for contralateral T2 Hsig. T1 Hsig showed bilateral abnormalities more frequently, whereas T2 Hsig could lateralize better hippocampal abnormalities. Overall, there were no differences when comparing T2 Hsig for the entire hippocampus, head, or tail only. However, there were some individual differences. T2 Hsig abnormalities involved the entire hippocampus in most patients but were restricted to the head in one patient and to the tail in another. CONCLUSIONS: This simplified method for the quantification of Hsig using NHI-Image is an efficient method for the identification and quantification of hippocampal abnormalities in patients with MTLE. The assessment of the entire extension of the hippocampal formation may provide important additional data, compared to T2 relaxometry maps limited to one segment of the hippocampus.

Adult↗
PubMed2003

The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression.

Real-time reverse transcriptase polymerase chain reaction (RT-PCR) is becoming a widely used method to quantify cytokines from cells, tissues, or tissue biopsies. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in a single step. Using TaqMan chemistry (Applied Biosystems, Foster City, CA) and the ABI Prism 7700 Sequence Detection System (Applied Biosystems), we validated a large panel of murine and human cytokines, as we as other factors playing a role in the immune system, such a chemokines and apoptotic markers. Although the method allows fast, sensitive, and accurate quantification, different control assays are necessary for the method to be reliable. By construction of complementary DNA (cDNA) plasmid clones, standard curves are generated that allow direct quantification of every unknown sample. Furthermore, the choice of a reliable housekeeping gene is very important. Finally, co-amplification of contaminating genomic DNA is avoided by designing sets of primers located in different exons or or intron-exon junctions. In conclusion, the real-time RT-PCF technique is very accurate and sensitive, allows high through put, and can be performed on very small samples. The development of real-time RT-PCR has resulted in an exponential increase in its use over the last couple of years, and the method has undoubtedly become the standard for quantifying cytokine patterns, clarifying many functional properties of immune cells and their associated diseases.

Animals↗
PubMed1992

[Analysis of the determinant factors for open heart surgery without blood transfusion by the quantification theory--possibility of application of maximum surgical blood order schedule for open heart surgery].

In order to complete operations without blood transfusion we have chosen means of preoperative autologous blood saving and intraoperative autotransfusion, but we have not always achieved our purpose. We examined 29 patients (13 patients without blood transfusion and 16 with blood transfusion) to analyze the determinant factors as to whether open heart surgery without blood transfusion may be indicated or not, according to the quantification theory (type II) and to examine the possibility to apply the maximum surgical blood order schedule (MSBOS) for the open heart surgery by the quantification theory (type I). The analysis of determinant factors revealed hematocrit (Ht) value before saving of blood (more than 40%) as the best contributor of possibility of non-blood transfusion surgery, followed by the amount of blood loss during operation (less than 600 ml), the amount of saving blood (more than 800 ml), body weight (less than 70 kg), calculated Ht value on the beginning of cardiopulmonary bypass (CPB) (more than 24%), CPB time (less than 120 minutes) and the amount of postoperative blood loss (less than 600 ml). The prospective using blood volume at the operation was precisely calculated by the values of 4 preoperative factors, that is, the amount of saving blood, calculated Ht value on the beginning of CPB, CPB time and body weight. Therefore it is important to increase the amount of preoperative saving blood and decrease the amount of surgical bleeding in order to perform operations without blood transfusion, and is possible to apply the MSBOS for the open heart surgery.

Adolescent↗
PubMed2003

[Genotyping and RNA quantification of hepatitis C virus. ].

Genotyping and quantification of hepatitis C virus is of importance in the assessment of prognosis and choice of treatment. We present the results from a study of 144 patients over two years in our hospital and point out the degree of uncertainty involved in the method of quantification. Doctors treating patients with hepatitis C should know the level of precision of the applied method.

Genotype↗
PubMed2003

Quantification of regional ventilation-perfusion ratios with PET.

UNLABELLED: The topographic matching of alveolar ventilation (V(A)) and perfusion (Q) is the main determinant of gas exchange efficiency of the lung. However, no pulmonary functional imaging technique has been shown to predict whole-lung gas exchange in health and disease. This study aims to present a PET-based method to estimate regional alveolar ventilation-to-perfusion ratios (V(A)/Q) predictive of arterial blood gases. METHODS: The method is based on the regional tracer kinetics of (13)N-nitrogen ((13)NN) after an intravenous bolus injection during a breath-hold period and subsequent washout from the lungs with resumption of breathing. The method takes into account the presence of inter- and intraregional nonuniformities at length scales smaller than the imaging spatial resolution. An algorithm used regional tracer washout to classify regional V(A)Q/ uniformity. Intraregional V(A)/Q mismatch in nonuniform regions was described with a 2-compartment model. Regional V(A)/Q estimates were combined into a whole-lung distribution of V(A)/Q ratios and were used to compute global arterial blood gases. The method was applied to 3-dimensional PET data from anesthetized and mechanically ventilated sheep before and after methacholine bronchoconstriction (n = 3) and pulmonary embolism (n = 3) and after saline lung lavage (n = 3). RESULTS: PET images revealed regional changes in ventilation and perfusion consistent with the different disease models. Quantification of the images using PET-derived V(A)Q/ distributions showed unimodal and narrow distributions in control conditions that became wider and unimodal after pulmonary embolism and saline lung lavage and bimodal after bronchoconstriction. Images of regional gas exchange allowed for visualization of regional gas exchange. Arterial blood gases estimated from the PET-based V(A)/Q distributions closely agreed with measured values (partial pressure of oxygen, arterial [PaO(2)]: r(2) = 0.97, P < 0.001; partial pressure of carbon dioxide, arterial [PaCO(2)]: r(2) = 0.96, P < 0.001). CONCLUSION: Tracer kinetics analysis of PET images after an intravenous injection of (13)NN provides a quantitative assessment of regional V(A)/Q heterogeneity including that corresponding to length scales smaller than the spatial resolution of the imaging method. Quantification of V(A)/Q mismatch obtained with the presented technique is directly related to severity of gas exchange impairment as determined by arterial blood gases.

Algorithms↗
PubMed1992

Contrast-enhanced CT scanning in vivo for the quantification of hepatic metastases from a human colonic cancer in the nude rat.

Hepatic metastases were induced in nude rats by intraportal injection of 2.5-5.0 x 10(6) cells from the human colonic cancer cell line LS 174 T. Quantification of tumour burden, expressed as relative metastatic area, was performed by contrast-enhanced computed tomography (CT) scanning in vivo (n = 14), contrast enhanced CT scanning post mortem (n = 21) and computer-based area calculation (CBAC) (n = 21). To determine the false-positive contribution to the estimated tumour burden by the evaluation procedures themselves, six rats without metastases were assessed. The quantification in the three different assessment groups was in close accordance in animals with an intermediate or extensive metastatic burden, but not in rats with a minor (< 4%) tumour burden. The results indicate that contrast-enhanced CT scanning can be used in this model to quantify hepatic metastases, except in animals with few and small lesions. Furthermore, the results suggest a potential for the assessment of therapeutic response by repeated contrast-enhanced CT scanning in vivo, as well as prospects for a corresponding evaluation in man.

Animals↗