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PubMed1995

Determination of triglycerides in lipoproteins separated by agarose gel electrophoresis.

We developed a simple method for the quantitation of triglycerides in electrophoretically separated lipoproteins by specific enzymatic staining. After electrophoresis, glycerol is liberated from triglycerides by the action of cholesterol esterase. Glycerol is oxidized by a sequence of enzymatic reactions. Due to the presence of triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase in the reaction mixture, two moles of the precipitating dye formazane are generated per mole glycerol. The relative amounts of alpha, pre-beta, and beta lipoproteins are determined by densitometric scanning at 570 nm. Absolute triglyceride concentrations of the respective lipoprotein fractions are calculated from total triglycerides. When tested with purified very low density lipoproteins, the electrophoresis assay was linear between 0.08 and 6.5 g/l pre-beta lipoprotein triglycerides. The intra-assay and inter-assay coefficients of variation were between 5.2% and 9.8%, and between 3.2% and 12.9%, respectively. Comparison of the electrophoresis method with a combined ultracentrifugation/precipitation method in 172 sera resulted in the following correlation coefficients: alpha lipoprotein versus high density lipoprotein triglycerides, r = 0.847; pre-beta lipoprotein versus very low density lipoprotein triglycerides, r = 0.989; beta lipoprotein versus low density lipoprotein triglycerides, r = 0.815. This method is easy to perform, and is a precise and accurate technique for the determination of lipoprotein triglycerides. It is the first reliable method that allows the direct quantification of LDL triglycerides without ultracentrifugation.

Analysis of Variance↗

PubMed2004

Simple and rapid liquid chromatography-tandem mass spectrometry confirmatory assay for determining amoxicillin and ampicillin in bovine tissues and milk.

A simple specific and rapid confirmatory method for determining the two amphoteric penicillins, that is, amoxicillin and ampicillin, in bovine muscle, liver, kidney, and milk is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography (LC)-tandem mass spectrometry. With this instrumentation, the selected reaction monitoring acquisition mode with two fragmentation reactions for each analyte was adopted. After acidification and filtration of the aqueous extracts, 25 microL of the tissue final extracts and 50 microL of the milk final extract were injected into the LC apparatus. Absolute recovery of the two analytes in any biological matrix at the 50 ppb level in tissues and the 4 ppb level in milk was 74-95% with relative standard deviations (RSDs) of no larger than 9%. When penicillin V was used as surrogate internal standard, relative recovery of the targeted compounds present in bovine tissues and milk at, respectively, 25 and 2 ppb levels ranged between 100 and 106% with RSDs of no larger than 11%. When fractionation of analytes by using a short chromatographic run was attempted, remarkable signal weakening for the two analytes was experienced. This effect was traced to polar endogenous coextractives eluted in the first part of the chromatographic run that interfered with the gas-phase ion formation of the two penicillins. Slowing the chromatographic run eliminated this unwelcome effect. Limits of quantification of the two analytes in bovine milk were estimated to be <1 ppb, whereas amoxicillin and ampicillin could be quantified in bovine tissues down to 3.1 and 0.8 ppb levels, respectively.

Amoxicillin↗

PubMed2003

Surrogate markers as a guide to evaluate response to antiretroviral therapy.

The development of an increasing number of antiretroviral agents has dramatically reduced HIV-associated morbidity and mortality. However, most of these drugs have been approved through clinical trials where only surrogate markers for clinical endpoints have been used. Ideally, a surrogate marker should be biologically plausible, predictive of disease progression and measurable by standardized assays. Historically, a number of candidate markers have been explored for monitoring the course of HIV infection and response to treatment. While the level of plasma HIV RNA and the absolute numbers of peripheral CD4+ T cells have eventually become the reference markers in clinical practice, several additional parameters are still being evaluated to improve our knowledge of the virus-host interaction, discriminate between apparently equivalent stages and further refine antiretroviral treatment. Advances in molecular methods and growing elucidation of HIV dynamics in vivo have made it possible to consider several molecular virologic parameters as candidate markers for treatment response, including intracellular levels of different HIV RNA species and amount of integrated and unintegrated HIV DNA. Much effort has been recently devoted to the definition of immunological parameters as prognostic markers. The abnormal activation induced by HIV on the immune system represents a major pathogenetic feature of HIV infection. Immune activation may be evaluated by the analysis of activation markers expressed on the cell membrane and by the quantification of soluble plasma molecules released by activated cells. Such markers of immune activation have an important prognostic significance in terms of disease progression and might be suitable for the monitoring and prognosis of antiretroviral therapies. In the late years, the possibility of extending potent antiretroviral therapies to developing countries has raised the need of simple, reliable and cost-effective tests to measure prognostic markers for disease evolution and assessment of therapy efficacy. This review summarizes the benefits and limits of reference and candidate surrogate markers and their integration for optimal antiretroviral therapy.

Animals↗

PubMed2005

Quantification of T-cell-mediated apoptosis in heterogeneous leukemia populations using four-color multiparameter flow cytometry.

BACKGROUND: The unique capacity of dendritic cells to present antigens to naive T cells is being increasingly utilized in cancer therapy. The efficacy of cell-based immunotherapy can be analyzed by determination of cytotoxic activity of T cells toward tumor cells in vitro. This study supplies a flow cytometric method to analyze T-cell-mediated cytotoxic activity toward heterogeneous leukemic cell populations at a single-cell level. METHODS: The fluorescent probe SYTO16 and the dead-cell dye 7-aminoactinomycine-D (7-AAD) were used to identify early and late stages of apoptosis in combination with leukemia cell-identifying markers. Determination of viable, apoptotic, and necrotic cells was performed by inclusion of fluorescent beads. RESULTS: In nine acute myeloid leukemia samples and three leukemic cell lines the use of SYTO16 next to the dead-cell marker 7-AAD significantly increased (P = 0.001) the sensitivity of the cytotoxicity assay as compared with single use of 7-AAD. Analysis of several effector-to-target ratios revealed the ability to determine dose-response effects. Enumeration of absolute numbers resulted in coefficients of variation of 4.1% and 8.4% for cell lines and leukemic samples, respectively. CONCLUSION: The presented flow cytometric cytotoxicity assay enables the study of T-cell-mediated apoptosis in a heterogenous leukemia population.

Apoptosis↗

PubMed1992

Quantification of adipose tissue by MRI: relationship with anthropometric variables.

This study had two objectives: 1) to establish magnetic resonance imaging (MRI) as a tool for measuring total and regional adipose tissue (AT) distribution in humans and 2) to assess the relationship between selected anthropometric variables and MRI-measured AT. Twenty-seven healthy men varying in age [40.8 +/- 14.5 (SD) yr], body mass index (28.5 +/- 4.8), and waist-to-hip ratio (WHR, 0.96 +/- 0.07) participated in the study. Total AT volume was determined using a linear interpolation of AT areas obtained on consecutive slices (n = 41) taken from head to toe (10-mm thickness, 50-mm centers). The mean change for repeated measures of total AT volume was 2.9% (range 0.9-4.3%). Large interindividual differences were observed for total AT volume (6.9-59.3 liters), subcutaneous AT (6.3-49.8 liters), and visceral AT (0.5-8.5 liters). Visceral AT represented 18.3% of the total AT. The single best predictor of total adiposity was waist circumference (R2 = 0.92). For visceral AT volume, WHR was the strongest anthropometric correlate (r = 0.85, P less than 0.01). When controlled for age and adiposity, however, WHR explained only 12% of the variation in absolute visceral AT and less than 1% of the variation in visceral-to-subcutaneous ratio. Age was a better predictor of visceral-to-subcutaneous ratio than level of adiposity or WHR. The results of this study demonstrate that MRI offers a reliable measure of regional and total AT distribution in humans and, thus, is of value as a research tool.

Adipose Tissue↗

PubMed2002

Electrothermal vaporisation ICP-mass spectrometry (ETV-ICP-MS) for the determination and speciation of trace elements in solid samples - A review of real-life applications from the author's lab.

The use of electrothermal vaporisation (ETV) from a graphite furnace as a means of sample introduction in inductively coupled plasma mass spectrometry (ICP-MS) permits the direct analysis of solid samples. A multi-step furnace temperature programme is used to separate the vaporisation of the target element(s) and of the matrix components from one another. Sometimes, a chemical modifier is used to enable a higher thermal pre-treatment temperature, by avoiding premature analyte losses (stabilisation) or promoting the selective volatilisation of matrix components. In almost all instances, accurate results can be obtained via external calibration or single standard addition using an aqueous standard solution. Absolute limits of detection are typically ~1 pg, which corresponds to 1 ng/g for a typical sample mass of 1 mg. Real-life applications carried out in the author's lab are used to illustrate the utility of this approach. These applications aim at trace element determination in industrial and environmental materials. The industrial materials analysed include different types of plastics - Carilon, polyethylene, poly(ethyleneterephtalate) and polyamide - and photo- and thermographic materials. As samples from environmental origin, plant material, animal tissue and sediments were investigated. Some applications aimed at a multi-element determination, while in other, the content of a single, but often challenging, element (e.g., Si or S) had to be measured. ETV-ICP-MS was also used in elemental speciation studies. Separation of Se-containing proteins was accomplished using polyacrylamide gel electrophoresis (PAGE). Subsequent quantification of the Se content in the protein spots was carried out using ETV-ICP-MS. As the volatilisation of methylmercury and inorganic mercury could be separated from one another with respect to time, no chromatographic or electrophoretic separation procedure was required, but ETV-ICP-MS as such sufficed for Hg speciation in fish tissue.

Mass Spectrometry↗

PubMedDate not supplied

Continuous urea monitoring in hemodialysis: a model approach to forecast dialytic performance. Results of a multicenter study.

BACKGROUND: A urea biosensor, inserted into the ultrafiltrate collection-line of paired filtration dialysis (PFD), not only allows on-line dialysis quantification, but also forecasts final (Cend) and 30 min equilibrated urea concentration (Ceq), the most reliable value for calculating dialysis efficiency. The urea biosensor processes plasma ultrafiltrate continuously, delivering a large amount of data to the computer, which estimates the parameters by a mathematical model, thus predicting the whole urea profile with rebound. METHODS: A multicenter randomized trial on 41 patients was conducted to ascertain the ability of a two-pool variable-volume urea model to forecast Cend and Ceq at 60 and 90 min after the start of dialysis. Two alternative dialytic treatments, A or B, were chosen, the latter being more efficient. Each treatment included six serial PFD. The accuracy of forecasting was evaluated through four indices based on forecast errors, calculated as the difference between observed and forecasted urea values: mean percent error (MPE) (%), mean absolute deviation (MAD) (mg/dl), mean absolute percent error (MAPE) (%) and root mean squared error (RMSE) (mg/dl). RESULTS: Forecasted urea concentrations were lower than those measured by the biosensor. MPE for Cend was negligible in A (+1.2%) and much higher in B (+7.2%); both values improved at 90 min, +1.0% and +5.8%, respectively. MAD for Cend was similar in both treatments and improved slightly at 90 min, ranging from 4.9 to 5.9 mg/dl. MPE for Ceq was +4% in A and and more than doubled in B (+11.5%); both values improved at 90 min, +3.7% and +9.7%, respectively. MAD for Ceq was 7.5 mg/dl in A and 8.5 mg/dl in B; both improved at 90 min, 6.7 and 7.4 m g/dl, respectively. The other indices, MAPE and RMSE, showed similar results. Comparison between the errors of the two treatments with analysis of variance (ANOVA) for repeated measures gave no significant results. CONCLUSIONS: Our model forecasts of urea concentrations were overall lower than the measured ones: the bias was negligible for A-Cend, greater for the A-Ceq and when the more efficient treatment B was used. The 60 min predictions improved at 90 min. The comparison between the prediction errors in the two treatments were not statistically significant. The recirculation measurement would probably reduce the bias if it were properly incorporated into the model.

Filtration↗

PubMed2001

Molecular quantification of viral load in plasma allows for fast and accurate prediction of response to therapy of Epstein-Barr virus-associated lymphoproliferative disease after allogeneic stem cell transplantation.

Epstein-Barr virus lymphoproliferative disease (EBV-LPD) following allogeneic stem cell transplantation (allo-SCT) has a poor prognosis. We used a sensitive real-time polymerase chain reaction (PCR) assay for quantitative detection of EBV-DNA in plasma and serially measured EBV-DNA levels to assess the response to treatment in allo-SCT recipients with EBV-LPD. Fourteen allo-SCT recipients with EBV-LPD who received a T cell-depleted (TCD) sibling (n = 5) or matched unrelated donor (n = 9) graft were monitored from the time of EBV-LPD diagnosis, during therapy and assessment of clinical response. Seven patients had complete responses of EBV-LPD to therapy, of whom 21% (3 out of 14) survived beyond 6 months from EBV-LPD diagnosis. Clinically responding patients showed a rapid decline of EBV-DNA plasma levels within 72 h from the start of therapy. In contrast, all clinical non-responders showed an increase of EBV-DNA levels. Absolute EBV-DNA levels at the time of EBV-LPD diagnosis did not predict for response, but the pattern of EBV-DNA levels within 72 h from the start of therapy (> 50% decrease versus increase) strongly predicted for clinical response (P = 0.001). Quantitative monitoring of EBV-DNA levels from the start of and during therapy for EBV-LPD rapidly and accurately predicts for response to therapy as early as within 72 h. It may thus provide a powerful tool to adjust and select treatment in individuals with EBV-LPD following allo-SCT.

Acute Disease↗

PubMed1996

Fish oil fatty acids and human platelets: dose-dependent decrease in dienoic and increase in trienoic thromboxane generation.

Dietary enrichment of membrane phospholipids with n-3 (fish-oil-derived) fatty acids has attracted attention as a putative therapeutic regimen for suppression of inflammatory and coagulatory events. Use of n-3 fatty-acid-enriched lipid infusions for parenteral nutrition results in micromolar concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DCHA) in the plasma-free fatty acid fraction. We investigated the influence of free EPA and DCHA on platelet thromboxane (Tx) A2 and A3 formation by using a recently developed high performance liquid chromatography-ELISA technique for separate quantification of the stable hydrolysis products TxB2 and TxB3. Washed human thrombocytes were incubated with free arachidonic acid (AA; 1 microM), A23187 (0.1 microM) or thrombin (5 U/mL) for stimulation; all regimens provoked large quantities of TxA2 in the absence of TxA3. Simultaneous admixture of free EPA or free DCHA to the incubation medium (concentration range, 0.01-50 microM) largely suppressed platelet TxA2 generation in response to all stimuli used in a dose-dependent manner. The effective concentration with 50% influence of arachidonic acid was 4.2 microM, whereas the inhibitory concentration with 50% effect of EPA and DCHA were both in the same order of magnitude but differed with the nature of the agonist (0.2-7 microM). Platelet (co-)incubation with EPA, but not DCHA, provoked dose-dependent synthesis of n-3-lipid-derived thromboxane: kinetics of formation and absolute quantities of TxA3 approximated 20% of the respective TxA2 data upon stimulation with AA. Both EPA and DCHA dose-dependently suppressed U46619-provoked platelet aggregation. We conclude that EPA and DCHA are potent competitive inhibitors of TxA2 generation by intact platelets, with EPA acting as poor substrate and DCHA being no substrate for the cyclooxygenase/thromboxane synthase complex. Enrichment of the plasma-free fatty acid fraction with n-3 lipids may offer a therapeutic regimen to suppress the synthesis of the potent proaggregatory and vasoconstrictory agent TxA2.

Animals↗

PubMed2006

Quantitative determination of absolute organohalogen concentrations in environmental samples by X-ray absorption spectroscopy.

An in situ procedure for quantifying total organic and inorganic Cl concentrations in environmental samples based on X-ray absorption near-edge structure (XANES) spectroscopy has been developed. Cl 1s XANES spectra reflect contributions from all Cl species present in a sample, providing a definitive measure of total Cl concentration in chemically heterogeneous samples. Spectral features near the Cl K-absorption edge provide detailed information about the bonding state of Cl, whereas the absolute fluorescence intensity of the spectra is directly proportional to total Cl concentration, allowing for simultaneous determination of Cl speciation and concentration in plant, soil, and natural water samples. Absolute Cl concentrations are obtained from Cl 1s XANES spectra using a series of Cl standards in a matrix of uniform bulk density. With the high sensitivity of synchrotron-based X-ray absorption spectroscopy, Cl concentration can be reliably measured down to the 5-10 ppm range in solid and liquid samples. Referencing the characteristic near-edge features of Cl in various model compounds, we can distinguish between inorganic chloride (Cl(inorg)) and organochlorine (Cl(org)), as well as between aliphatic Cl(org) and aromatic Cl(org), with uncertainties in the range of approximately 6%. In addition, total organic and inorganic Br concentrations in sediment samples are quantified using a combination of Br 1s XANES and X-ray fluorescence (XRF) spectroscopy. Br concentration is detected down to approximately 1 ppm by XRF, and Br 1s XANES spectra allow quantification of the Br(inorg) and Br(org) fractions. These procedures provide nondestructive, element-specific techniques for quantification of Cl and Br concentrations that preclude extensive sample preparation.

Bromine↗

PubMed1975

Quantification of triglyceride transport in blood plasma: a critical analysis.

Reliable and precise quantification of endogenous triglyceride transport in man has not been possible with simple means to date. Direct measurement of net splanchnic secretion of triglyceride fatty acids (TGFA) in very low density lipoproteins (VLDL) provides the must unambiguous information, but precision is low. Coupling infusion of labeled fatty acid with sampling of arterial and hepatic venous blood increases precision; however, the contribution of precursors other than plasma free fatty acids (FFA) must be assessed. Measurement of the rate of hydrolysis of plasma triglycerides after displacing lipases into the blood with heparin holds promise as a simple, nonisotopic method, but it has not been carefully validated and heparin itself alters FFA and triglyceride transport. Multicompartmental analysis following pulse injection of labeled fatty acid offers a practical approach, but uncertainties about the number and location of interacting compartments have made it impossible to determine an absolute value for transport. Reinjection of biologically labeled plasma VLDL is impractical for large scale use, and validity of this approach remains uncertain because of heterogeneity of VLDL-triglycerides and their complex metabolic behavior. Methods to label VLDL-triglycerides in vitro deserve more study as does labeling of other components, such as the B-apoprotein. Such approaches will require rigorous comparison with biologically labeled material as well as careful assessment of alterations in kinetic behavior that may occur when VLDL are separated from blood plasma.

Adult↗

PubMed1999

Analysis of pristanic acid beta-oxidation intermediates in plasma from healthy controls and patients affected with peroxisomal disorders by stable isotope dilution gas chromatography mass spectrometry.

In this paper we report the development of highly sensitive, selective, and accurate stable isotope dilution gas chromatography negative chemical ionization mass spectrometry (GC-NCI-MS) methods for quantification of peroxisomal beta-oxidation intermediates of pristanic acid in human plasma: 2,3-pristenic acid, 3-hydroxypristanic acid, and 3-ketopristanic acid. The carboxylic groups of the intermediates were converted into pentafluorobenzyl esters, whereas hydroxyl groups were acetylated and ketogroups were methoximized. Hereafter, the samples were subjected to clean-up by high performance liquid chromatography. Analyses were performed by selected monitoring of the carboxylate anions of the derivatives. Control values of all three metabolites were established (2,3-pristenic acid: 2-48 nm, 3-hydroxypristanic acid: 0.02-0.81 nm, 3-ketopristanic acid: 0.07-1.45 nm). A correlation between the concentrations of pristanic acid and its intermediates in plasma was found. The diagnostic value of the methods is illustrated by measurements of the intermediates in plasma from patients with peroxisomal disorders. It is shown that in generalized peroxisomal disorders, the absolute concentrations of 2,3-pristenic acid, 3-hydroxypristanic acid, and 3-ketopristanic acid were comparable to those in the controls, whereas relative to the pristanic acid concentrations these intermediates were significantly decreased. In bifunctional protein deficiency, elevated levels of 2,3-pristenic acid and 3-hydroxypristanic acid were found. 3-Ketopristanic acid, although within the normal range, was relatively low when compared to the high pristanic acid levels in these patients.-Verhoeven, N. M., D. S. M. Schor, E. A. Struys, E. E. W. Jansen, H. J. ten Brink, R. J. A. Wanders, and C. Jakobs. Analysis of pristanic acid beta-oxidation intermediates in plasma from healthy controls and patients affected with peroxisomal disorders by stable isotope dilution gas chromatography mass spectrometry.

Diagnosis, Differential↗

PubMed1991

Identification and quantification of aberrant crypt foci and microadenomas in the human colon.

The objective of the present study was to determine whether aberrant crypt foci (ACF) similar to those observed in the colons of experimental animals exposed to colon carcinogens could be identified and quantified in the human colon. Twenty-seven colon resections from patients affected by familial adenomatous polyposis (FAP, five cases), colorectal cancer (CRC, 12 cases), and benign diseases of the large bowel (BD, 10 cases) were collected from a pathology repository or immediately after operation. Ten or more 1-cm2 formalin-fixed, methylene-blue--stained samples of colonic mucosa from each colon were scored under light microscopy for ACF. The number of ACF per cm2 and the number of crypts per ACF for each colon were calculated. The average number of ACF per cm2 in the FAP group (20 +/- 19, mean +/- SD) was significantly higher (P less than 0.01) than those of the CRC (0.37 +/- 0.41) and BD (0.18 +/- 0.35) groups. At least one ACF was found in every colon resection from CRC patients and in six out of 10 colon resections from the BD group. The average number of crypts per ACF ranged from five to 35 with absolute values from 1 to over 100. Fifty-five histologic specimens, 43 with ACF of various size and 12 without, were prepared by sectioning the colon parallel to the mucosal surface. There was a close correlation between the number of crypts per ACF in each specimen as scored by methylene-blue and histologic examination. Twenty-six aberrant crypt foci displayed dysplasia as evident by histologic analysis. In these instances we feel the term microadenoma is appropriate and, using this unique approach of examining the human colon, they can be easily identified and quantified. These lesions may well be precursors for adenomatous polyps and colorectal cancer.

Adenoma↗

PubMed2006

[Development of software for three-dimensional reconstruction and automatic quantification of intravascular ultrasound images. Initial experience].

INTRODUCTION AND OBJECTIVES: Quantification of intravascular ultrasound (IVUS) images is essential in ischemic heart disease and interventional cardiology. Manual analysis is very slow and expensive. We describe an automated computerized method of analysis that requires only minimal initial input from a specialist. METHODS: This study was carried out by interventional cardiologists and biomedical engineers working in close collaboration. We developed software in which it was necessary only to identify the media-adventitia boundary in a few images taken from the whole sequence. A three-dimensional reconstruction was then generated from each sequence, from which measurements of areas and volumes could be derived automatically. In total, 2300 randomly selected images from video sequences of 11 patients were analyzed. RESULTS: Results obtained using the proposed method differed only minimally from those obtained with the manual method: for vessel area measurements, the variability was 0.08 (0.07) (mean absolute error [standard deviation] normalized to the actual value; this corresponds to an error of 0.08 mm(2) per mm(2) of vessel area); for lumen area, 0.11 (0.11) (normalized), and for plaque volume, 0.5 (0.3) (normalized). Regions with severe lesions (<4 mm(2)) were correctly identified in more than 90% of cases. Specialist time needed for each reconstruction was 10 (8) minutes (vs 60 [10] minutes for manual analysis; P< .0001). CONCLUSIONS: The computerized method used dramatically reduced the time and effort needed for IVUS sequence analysis, and the automated measurements obtained were very promising.

Coronary Artery Disease↗

PubMed1996

Quantification of MRI lesion load in multiple sclerosis: a comparison of three computer-assisted techniques.

Several computer-assisted techniques for measuring multiple sclerosis lesion load on MR images have been developed to provide a quantitative and sensitive means for monitoring disease activity, particularly in the context of treatment trials. We have evaluated three techniques: manual outlining (similar to that of the North American interferon beta-1b trial), semiautomated lesion contouring (local lesion based threshold), and intensity-based thresholding for the whole brain. Contiguous, 5 mm-thick, axial, T2-weighted images of the brain were obtained on a 1.5T MR imager in eight patients with clinically definite multiple sclerosis. Analyses of the scans were performed twice, independently by three operators, using the three different techniques. The coefficient of variation of the measurement techniques was: (a) intrarater precision, 9.0 +/- 5.2 (mean +/- SD) (range 0.4-18.5) for the manual outlining, 2.5 +/- 2.1 (0.1-7.7) for the contour technique, and 7.5 +/- 6.9 (0.2-22.0) for the global threshold technique; (b) interrater precision, 11.0 +/- 5.8 (4.9-21.7) for the manual outlining, 4.5 +/- 1.6 (1.8-6.6) for the contour technique, and 11.4 +/- 4.9 (2.8-19.2) for the global threshold technique (0.0 = perfect precision). The absolute lesion loads measured were very similar using the manual outlining and the contour techniques but were significantly smaller using the global threshold technique. We conclude that the contour technique is a promising tool for use in treatment trials. Further studies are needed to assess sensitivity to changes in lesion load over time.

Brain↗

PubMed1998

Quantification of myosin heavy chain mRNA in somatic and branchial arch muscles using competitive PCR.

The purpose of this study was to quantify the type and amount of myosin heavy chain (MHC) mRNA within muscles of different developmental origins to determine whether the regulation of gene expression is comparable. Seven MHC isoforms were analyzed in rat adult limb (extensor digitorum longus, tibialis anterior, and soleus) and nonlimb (extraocular, thyroarytenoid, diaphragm, and masseter) muscles using a competitive PCR assay. An exogenous template that included oligonucleotide sequences specific for seven rat sarcomeric MHC isoforms (beta-cardiac, 2A, 2X, 2B, extraocular, embryonic, and neonatal) as well as beta-actin was constructed and used as the competitor. Only the extraocular muscle contained all seven isoforms. All seven muscles contained type 2A and type 2X MHC transcripts in varying percentages. As expected, the soleus muscle contained primarily beta-cardiac MHC (87.8 +/- 2.6%). Extraocular MHC was found only in the extraocular and thyroarytenoid muscles and in relatively small proportions (7.4 +/- 1.5% and 4.0 +/- 0.7%, respectively). Neonatal MHC was identified in extraocular (7.9 +/- 0. 3%), thyroarytenoid (4.4 +/- 0.4%), and masseter (1.0 +/- 0.2%) muscles, and embryonic MHC was identified both in extraocular (1.2 +/- 0.5%) and, unexpectedly, in soleus (0.6 +/- 0.1%) muscles. Absolute MHC mRNA mass was greatest in the masseter (106 pg/0.5 microg RNA) and least for the tibialis anterior (64 pg/0.5 microg RNA). These values suggest that MHC mRNA represents from 4 to 17% of the total mRNA pool in various skeletal muscles. Differences in MHC profile between somatic and branchial arch muscles suggest that the developmental origin of a muscle may, at least in part, be responsible for the MHC expression program that is implemented in the adult. An inverse relationship between the expression of beta-cardiac and type 2B MHC transcripts across muscles was noted, suggesting that the expression of these two isoforms may be reciprocally regulated.

Aging↗

PubMed1991

Quantification of antibodies to human growth hormone by high-performance protein G affinity chromatography with fluorescence detection.

The technique of high-performance affinity chromatography (HPAC) is applied to the quantitative determination of antibodies to human growth hormone (hGH) in serum from patients. An affinity column consisting of covalently immobilized protein G on a rigid support is used to capture the antibodies. Texas Red labeled hGH (hGH-TR) is used as a fluorescence probe for detecting the anti-hGH antibodies. Calibration curves are established by using a well-characterized monoclonal antibody to hGH (GHC101). The minimum detectable concentration (MDC) of anti-hGH antibody in serum is 250 ng/mL (this represents 10 ng of anti-hGH injected onto the protein G column). Analytical recoveries are 92-110% for seven replicates with 250-4000 ng/mL of GHC101. A precision of 15% relative standard deviation (RSD) can be achieved at the MDC. The precision is better above the detection limit. The linear dynamic range of the method is approximately 2 orders of magnitude. The total fluorescence recovery from the affinity column is greater than or equal to 96%. Sample analysis times are on the order of 20 min. The HPAC technique gives results in absolute units of concentration that correlate well with binding capacity values determined by radioimmunoassay.

Antibodies↗

PubMed1997

Analysis of single and double-stained alveolar macrophages by flow cytometry.

The quantification of cell surface antigens on human alveolar macrophages using flow cytometry is complicated by strong autofluorescence which varies with cell size and granularity. We report here a new method for overcoming the analytical problems caused by autofluorescence. After positioning the unstained cells along the 0, 0; 10(4), 10(4) diagonal on all three fluorescence dot-plots (FL1 vs. FL2; FL2 vs. FL3; FL1 vs. FL3) of a single-laser flow cytometer (excitation wavelength at 488 nm) and adjusting compensation so that the reference FL3 channel profile is not changed by PE-staining, the cell population on the FL3 histogram is arbitrarily classified into subpopulations having similar autofluorescence intensity. These are subsequently back-gated onto the FL1 vs. FL2 dot-plot and separately analyzed. The percentage of stained cells in the whole population is then calculated on the basis of absolute numbers or as the weighted mean of all the subpopulations. This approach permits the analysis not only of single-stained but also double-stained human alveolar macrophages.

Antigens, Surface↗