Cyclosporin monitoring in Australasia: 2002 update of consensus guidelines

Therapeutic drug monitoring of cyclosporin (CsA) has been established as part of the routine clinical treatment of patients following organ transplantation for more than 20 years, and based on contemporary knowledge, many consensus guidelines have been published to assist clinics and laboratories attain optimal strategies for patient care. This article addresses the newer directions in CsA monitoring, with particular reference to the Australasian situation that has evolved since the 1993 Australasian guideline (1). These changes have included the introduction of alternative assay methodologies, changed CsA formulation from Sandimmun to Neoral throughout Australasia, and alternatives to trough concentration (C0) monitoring, especially 2-hour concentration (C2) monitoring and associated validated dilution protocols to accurately quantitate the higher whole blood CsA concentrations. The revision was prepared following a recent survey of all Australasian CsA-monitoring laboratories (2) where discordant practices were evident.

New fluorescence parameters for monitoring photosynthesis in plants - 1. The effect of illumination on the fluorescence parameters of the JIP-test

Chlorophyll fluorescence measurements have a wide range of applications from basic understanding of photosynthesis functioning to plant environmental stress responses and direct assessments of plant health. The measured signal is the fluorescence intensity (expressed in relative units) and the most meaningful data are derived from the time dependent increase in fluorescence intensity achieved upon application of continuous bright light to a previously dark adapted sample. The fluorescence response changes over time and is termed the Kautsky curve or chlorophyll fluorescence transient. Recently, Strasser and Strasser (1995) formulated a group of fluorescence parameters, called the JIP-test, that quantify the stepwise flow of energy through Photosystem II, using input data from the fluorescence transient. The purpose of this study was to establish relationships between the biochemical reactions occurring in PS II and specific JIP-test parameters. This was approached using isolated systems that facilitated the addition of modifying agents, a PS II electron transport inhibitor, an electron acceptor and an uncoupler, whose effects on PS II activity are well documented in the literature. The alteration to PS II activity caused by each of these compounds could then be monitored through the JIP-test parameters and compared and contrasted with the literature. The known alteration in PS II activity of Chenopodium album atrazine resistant and sensitive biotypes was also used to gauge the effectiveness and sensitivity of the JIP-test. The information gained from the in vitro study was successfully applied to an in situ study. This is the first in a series of four papers. It shows that the trapping parameters of the JIP-test were most affected by illumination and that the reduction in trapping had a run-on effect to inhibit electron transport. When irradiance exposure proceeded to photoinhibition, the electron transport probability parameter was greatly reduced and dissipation significantly increased. These results illustrate the advantage of monitoring a number of fluorescence parameters over the use of just one, which is often the case when the F-V/F-M ratio is used.

Fluorescence Microscopy Imaging Denoising with Log-Euclidean Priors and Photobleaching Compensation

Fluorescent protein microscopy imaging is nowadays one of the most important tools in biomedical research. However, the resulting images present a low signal to noise ratio and a time intensity decay due to the photobleaching effect. This phenomenon is a consequence of the decreasing on the radiation emission efficiency of the tagging protein. This occurs because the fluorophore permanently loses its ability to fluoresce, due to photochemical reactions induced by the incident light. The Poisson multiplicative noise that corrupts these images, in addition with its quality degradation due to photobleaching, make long time biological observation processes very difficult. In this paper a denoising algorithm for Poisson data, where the photobleaching effect is explicitly taken into account, is described. The algorithm is designed in a Bayesian framework where the data fidelity term models the Poisson noise generation process as well as the exponential intensity decay caused by the photobleaching. The prior term is conceived with Gibbs priors and log-Euclidean potential functions, suitable to cope with the positivity constrained nature of the parameters to be estimated. Monte Carlo tests with synthetic data are presented to characterize the performance of the algorithm. One example with real data is included to illustrate its application.

Convex Total Variation Denoising of Poisson Fluorescence Confocal Images With Anisotropic Filtering

Fluorescence confocal microscopy (FCM) is now one of the most important tools in biomedicine research. In fact, it makes it possible to accurately study the dynamic processes occurring inside the cell and its nucleus by following the motion of fluorescent molecules over time. Due to the small amount of acquired radiation and the huge optical and electronics amplification, the FCM images are usually corrupted by a severe type of Poisson noise. This noise may be even more damaging when very low intensity incident radiation is used to avoid phototoxicity. In this paper, a Bayesian algorithm is proposed to remove the Poisson intensity dependent noise corrupting the FCM image sequences. The observations are organized in a 3-D tensor where each plane is one of the images acquired along the time of a cell nucleus using the fluorescence loss in photobleaching (FLIP) technique. The method removes simultaneously the noise by considering different spatial and temporal correlations. This is accomplished by using an anisotropic 3-D filter that may be separately tuned in space and in time dimensions. Tests using synthetic and real data are described and presented to illustrate the application of the algorithm. A comparison with several state-of-the-art algorithms is also presented.

Detection of Change in Fluorescence Between Reactive Cyan and the Yellow Fluorophores Using a-SiC:H Multilayer Transducers

The transducer consists of a semiconductor device based on two stacked -i-n heterostructures that were designed to detect the emissions of the fluorescence resonance energy transfer between fluorophores in the cyan (470 nm) and yellow (588 nm) range of the spectrum. This research represents a preliminary study on the use of such wavelength-sensitive devices as photodetectors for this kind of application. The device was characterized through optoelectronic measurements concerning spectral response measurements under different electrical and optical biasing conditions. To simulate the fluorescence resonance energy transfer (FRET) pairs, a chromatic time-dependent combination of cyan and yellow wavelengths was applied to the device. The generated photocurrent was measured under reverse and forward bias to read out the output photocurrent signal. A different wavelength-biasing light was also superimposed. Results show that under reverse bias, the photocurrent signal presents four separate levels, each one assigned to the different wavelength combinations of the FRET pairs. If a blue background is superimposed, the yellow channel is enhanced and the cyan suppressed, while under red irradiation, the opposite behavior occurs. So, under suitable biasing light, the transducer is able to detect separately the cyan and yellow fluorescence pairs. An electrical model, supported by a numerical simulation, supports the transduction mechanism of the device.

Inherently chiral calix[4]arenes with planar chirality: two new entries to the family

The synthesis of two new inherently chiral calix[4]arenes (ICCs, 1 and 2), endowed with electron-rich concave surfaces, has been achieved through the desymmetrization of a lower rim distal-bridged oxacyclophane (OCP) macrocycle. The new highly emissive ICCs were resolved by chiral HPLC, and the enantiomeric nature of the isolated antipodes proved by electronic circular dichroism (CD). Using time-dependent density functional calculations of CD spectra, their absolute configurations were established. NMR studies with (S)-Pirkle's alcohol unequivocally showed that the host-guest interactions occur in the chiral pocket comprehending the calix-OCP exo cavities and the carbazole moieties.

A potentiometric magnetic immunoassay for rapid detection of Salmonella typhimurium

Potentiometric detection with homemade polymeric membrane microelectrodes was coupled to a magnetic sandwich immunoassay for Salmonella typhimurium determination. Cadmium and sodium ion selective electrodes were used respectively as indicator and pseudo-reference electrodes and were prepared in pipette tips to allow potentiometric measurements in microliter sample volumes. In the proposed method, the concentration of S. typhimurium was proportional to the amount of cadmium released upon dissolution of a CdS nanoparticle labeled to the secondary detection antibody. The limit of detection was 2 cells per 100 μL. The immunomagnetic assay with potentiometric detection is suitable for sensitive and rapid (average total time per assay of 75 minutes) detection of S. typhimurium in milk samples. The proposed method is easy to perform, safe, sensitive, and low cost and has potential for in situ analysis.

Use of a fluorescence-based approach to assess short-term responses of the alga Pseudokirchneriella subcapitata to metal stress

This work explores the use of fluorescent probes to evaluate the responses of the green alga Pseudokirchneriella subcapitata to the action of three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II) for a short time (6 h). The toxic effect of the metals on algal cells was monitored using the fluorochromes SYTOX Green (SG, membrane integrity), fluorescein diacetate (FDA, esterase activity) and rhodamine 123 (Rh123, mitochondrial membrane potential). The impact of metals on chlorophyll a (Chl a) autofluorescence was also evaluated. Esterase activity was the most sensitive parameter. At the concentrations studied, all metals induced the loss of esterase activity. SG could be used to effectively detect the loss of membrane integrity in algal cells exposed to 0.32 or 1.3 μmol L−1 Cu(II). Rh123 revealed a decrease in the mitochondrial membrane potential of algal cells exposed to 0.32 and 1.3 μmol L−1 Cu(II), indicating that mitochondrial activity was compromised. Chl a autofluorescence was also affected by the presence of Cr(VI) and Cu(II), suggesting perturbation of photosynthesis. In conclusion, the fluorescence-based approach was useful for detecting the disturbance of specific cellular characteristics. Fluorescent probes are a useful diagnostic tool for the assessment of the impact of toxicants on specific targets of P. subcapitata algal cells.

HTLV-1/2 seroprevalence and coinfection rate in Brazilian first-time blood donors: an 11-year follow-up

The seroprevalence and geographic distribution of HTLV-1/2 among blood donors are extremely important to transfusion services. We evaluated the seroprevalence of HTLV-1/2 infection among first-time blood donor candidates in Ribeirão Preto city and region. From January 2000 to December 2010, 1,038,489 blood donations were obtained and 301,470 were first-time blood donations. All samples were screened with serological tests for HTLV-1/2 using enzyme immunoassay (EIA). In addition, the frequency of coinfection with hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), Chagas disease (CD) and syphilis was also determined. In-house PCR was used as confirmatory test for HTLV-1/2. A total of 296 (0.1%) first-time donors were serologically reactive for HTLV-1/2. Confirmatory PCR of 63 samples showed that 28 were HTLV-1 positive, 13 HTLV-2 positive, 19 negative and three indeterminate. Regarding HTLV coinfection rates, the most prevalent was with HBV (51.3%) and HCV (35.9%), but coinfection with HIV, CD and syphilis was also detected. The real number of HTLV-infected individual and coinfection rate in the population is underestimated and epidemiological studies like ours are very informative.

RS virus diagnosis: comparison of isolation, immunofluorescence and enzyme immunoassay

Two techniques for rapid diagnosis, immunofluorescence (IFAT) and enzyme immunoassay (EIA), have been compared with virus isolaion in tissue culture for the detection of respiratory syncytial virus (RSV) in specimens of nasopharyngeal secretions. The specimens were obtained from children under five years of age suffering from acute respiratory iliness, during a period of six months from January to June 1982. Of 471 specimens examined 54 (11.5%) were positive by virus isolation and 180 (38.2%) were positive by immunofluorescence. The bacterial contamination of inoculated tissue cultures unfortunately prevented the isolation of virus from many samples. Specimens from 216 children were tested to compare enzyme immunoassay and immunofluorescence. Of these 60 (27%) were positive by EIA and 121 (56%) were positive by IFAT. Our results suggest that the EIA technique although highly specific is rather insensitive. This may be because by the time these tests were done the originl nasopharyngeal secretions were considerably diluted and contained more mucus fragments than the call suspension used for IFAT. Of the three techniques, IFAT gives the best results although EIA may be useful where IFAT is not possible.

Rapid high-performance liquid chromatographic determination with fluorescence detection of furosemide in human body fluids and its confirmation by gas chromatography-mass spectrometry.

Furosemide (FD: Lasix) is a loop diuretic which strongly increases both urine flow and electrolyte urinary excretion. Healthy volunteers were administered 40 mg orally (dissolved in water) and concentrations of FD were determined in serum and urine for up to 6 h for eight subjects, who absorbed water at a rate of 400 ml/h. Quantification was performed by HPLC with fluorescence detection (excitation at 233 nm, emission at 389 nm) with a limit of detection of 5 ng/ml for a 300-microliters sample. The elution of FD was completed within 4 min using a gradient of acetonitrile concentration rising from 30 to 50% in 0.08 M phosphoric acid. The delay to the peak serum concentration ranged from 60 to 120 min. FD was still easily measurable in the sera from all subjects 6 h after administration. In urine, the excretion rates reached their maximum between 1 and 3 h. The total amount of FD excreted in the urine averaged 11.2 mg (range 7.6-14.0 mg), with a mean urine volume of 3024 ml (range 2620-3596 ml). Moreover, the urine density was lower than 1.010 (recommended as an upper limit in doping analysis to screen diuretics) only for 2 h. An additional volunteer was administered 40 mg of FD and his urine was collected over a longer period. FD was still detectable 48 h after intake. Gas chromatography-mass spectrometry with different types of ionization was used to confirm the occurrence of FD after permethylation of the extract. Negative-ion chemical ionization, with ammonia as reactant gas, was found to be the most sensitive method of detection.

Separation of free amino acids in human plasma by capillary electrophoresis with laser induced fluorescence: potential for emergency diagnosis of inborn errors of metabolism.

Free amino acids (AAs) in human plasma are derivatized with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) and analyzed by capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The labeling procedure is significantly improved over results reported previously. Derivatization can be completed in 40 min, with concentrations as low as 4 x 10(-8) M successfully labeled in favourable cases. Twenty-nine AAs (including 2 internal standards) are identified and can be reproducibly separated in 70 min. Migration time RSD values for 23 of these AAs were calculated and found in the range from 0.5 to 4%. The rapid derivatization procedure and the resolution obtained in the separation are sufficient for a semi-quantitative, emergency diagnosis of several inborn errors of metabolism (IEM). Amino acid profiles for both normal donor plasma samples and plasma samples of patients suffering from phenylketonuria, tyrosinemia, maple syrup urinary disease, hyperornithinemia, and citrullinemia are studied.

Noninvasive imaging of 5-HT3 receptor trafficking in live cells: from biosynthesis to endocytosis.

Sequential stages in the life cycle of the ionotropic 5-HT(3) receptor (5-HT(3)R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT(3)R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT(3)R-specific antagonist. After novel protein synthesis was chemically blocked, the 5-HT(3) R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quantitatively to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT(3) receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 mum/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT(3) receptors was observed within minutes after exposure to an extracellular agonist. Our orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking.

Serodiagnosis of Trypanosoma cruzi Infection Using the New Particle Gel Immunoassay - ID-PaGIA Chagas

The ID-Chagas test is a particle gel immunoassay (PaGIA). Red coloured particles are sensitised with three different synthetic peptides representing antigen sequences of Trypanosoma cruzi: Ag2, TcD and TcE. When these particles are mixed with serum containing specific antibodies, they agglutinate. The reaction mixture is centrifuged through a gel filtration matrix allowing free agglutinated particles to remain trapped on the top or distributed within the gel. The result can be read visually. In order to investigate the ability of the ID-PaGIA to discriminate negative and positive sera, 111 negative and 119 positive, collected in four different Brazilian institutions, were tested by each of the participants. All sera were previously classified as positive or negative according to results obtained with three conventional tests (indirect immunofluorescence, indirect hemaglutination, and enzime linked immunosorbent assay). Sensitivity rates of ID-PaGIA varied from 95.7% to 97.4% with mean sensitivity of 96.8% and specificity rates varied from 93.8 to 98.8% with mean specificity of 94.6%. The overall Kappa test was 0.94. The assay presents as advantages the simplicity of operation and the reaction time of 20 min. In this study, ID-PaGIA showed to be highly sensitive and specific.

Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry. II: Confirmatory analysis.

For doping control, analyses of samples are generally achieved in two steps: a rapid screening and, in the case of a positive result, a confirmatory analysis. A two-step methodology based on ultra-high-pressure liquid chromatography coupled to a quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was developed to screen and confirm 103 doping agents from various classes (e.g., beta-blockers, stimulants, diuretics, and narcotics). The screening method was presented in a previous article as part I (i.e., Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry. Part I: screening analysis). For the confirmatory method, basic, neutral and acidic compounds were extracted by a dedicated solid-phase extraction (SPE) in a 96-well plate format and detected by MS in the tandem mode to obtain precursor and characteristic product ions. The mass accuracy and the elemental composition of precursor and product ions were used for compound identification. After validation including matrix effect determination, the method was considered reliable to confirm suspect results without ambiguity according to the positivity criteria established by the World Anti-Doping Agency (WADA). Moreover, an isocratic method was developed to separate ephedrine from its isomer pseudoephedrine and cathine from phenylpropanolamine in a single run, what allowed their direct quantification in urine.