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PubMed1986

Extraction of nuclei from selected regions in paraffin-embedded tissue.

A method for the extraction of nuclei from selected regions in paraffin-embedded tissue is described. Fifty-micrometer sections are cut, dewaxed, and rehydrated. For the final handling, the sections are manually transferred from one tray to another. The sections are put on a slide under a dissection microscope and the region of interest is isolated by scraping off the irrelevant region with a scalpel. An optimal number of single nuclei is obtained by incubation in a protease solution with intermediate syringing. The nuclei are washed and can be used for flow cytometry. Resuspension of the nuclei in foetal calf serum and cytocentrifugation results in preparations suited for image analysis. DNA cytometric measurements of nuclei in a carcinoma in situ and an invasive carcinoma region in breast tissue present in the same tissue block and in a severe dysplasia/carcinoma in situ (CIN III) region of the cervix are presented.

Breast Neoplasms↗
PubMed2005

Analytical approaches to the determination of simple biophenols in forest trees such as Acer (maple), Betula (birch), Coniferus, Eucalyptus, Juniperus (cedar), Picea (spruce) and Quercus (oak).

Analytical methods are reviewed for the determination of simple biophenols in forest trees such as Acer (maple), Betula (birch), Coniferus, Eucalyptus, Juniperus (cedar), Picea (spruce) and Quercus (oak). Data are limited but nevertheless clearly establish the critical importance of sample preparation and pre-treatment in the analysis. For example, drying methods invariably reduce the recovery of biophenols and this is illustrated by data for birch leaves where flavonoid glycosides were determined as 12.3 +/- 0.44 mg g(-1) in fresh leaves but 9.7 +/- 0.35 mg g(-1) in air-dried samples (data expressed as dry weight). Diverse sample handling procedures have been employed for recovery of biophenols. The range of biophenols and diversity of sample types precludes general procedural recommendations. Caution is necessary in selecting appropriate procedures as the high reactivity of these compounds complicates their analysis. Moreover, our experience suggests that their reactivity is very dependent on the matrix. The actual measurement is less contentious and high performance separation methods particularly liquid chromatography dominate analyses whilst coupled techniques involving electrospray ionization are becoming routine particularly for qualitative applications. Quantitative data are still the exception and are summarized for representative species that dominate the forest canopy of various habitats. Reported concentrations for simple phenols range from trace level (<0.1 microg g(-1)) to in excess of 500 microg g(-1) depending on a range of factors. Plant tissue is one of these variables but various biotic and abiotic processes such as stress are also important considerations.

Chromatography, High Pressure Liquid↗
PubMed2006

Microfluidic handling of PCR solution and DNA amplification on a reaction chamber array biochip.

A microfluidic biochip for conducting an array of polymerase chain reaction (PCR) simultaneously was fabricated to understand the microfluidic loading process of PCR solution into microfabricated glass reaction chambers. The geometrical factors of the microfluidic structure, including the shape and depth of the microchamber, shape and size of the microchannels were investigated on the formation of air bubbles trapped within the microchamber during the PCR solution loading process. Furthermore, the effects of surface properties of the microfluidic structure, including hydrophilicity of the microchamber and inlet channel, and hydrophobicity of the outlet channel, on the loading of PCR solution, especially on the formation of air bubbles were studied. As a result, the surface wetting property of the microchamber was found to be the main reason for the formation of the air bubbles inside the microchamber during the loading of PCR solution in the biochips. A solution to avoid the air trapping has been proposed and investigated.

DNA↗
PubMed2005

Effect of sample handling on analysis of cytokine responses to Mycobacterium tuberculosis in clinical samples using ELISA, ELISPOT and quantitative PCR.

Measuring cytokine responses to infection has proven to be invaluable for the understanding of immunity to tuberculosis in the laboratory. However, far less data are available from studies in humans and these have often produced conflicting results. Here we describe a comprehensive multi-center comparison of the most commonly used protocols for cytokine analysis: ELISA, ELISPOT and RT-PCR, in cohorts of TB patients, their household contacts and community controls. In particular, we have studied the effect on these protocols of conditions that commonly prevail in field studies, such as delays between sample collection and analysis, or different source material, such as whole blood or frozen PBMC. The results clearly show that while there is good correlation between the methods under optimal conditions, each method has strengths and weaknesses that render them more or less suitable for particular types of analyses. Researchers should carefully consider these factors when planning human field studies.

Adolescent↗
PubMed1991

[Fundamental studies on measurement of the eosinophil cationic protein in sputum from patients with bronchial asthma].

One of the characteristics of patients with bronchial asthma is activation of eosinophils in the bronchi, which can be evaluated by measuring concentrations of the cationic granule proteins. Sputum seems to be the best material to evaluate the activation of eosinophils in the bronchi because it can be collected easily from the same patient every day even if he (she) has an asthmatic attack. Recently it has become possible to measure the concentration of the eosinophil cationic protein (ECP) thanks to the availability of the ECP RIA kit. However no fundamental studies have been carried out on the handling of sputum samples, and whether only ECP released into sputum is measured. We prepared supernatants of sputum samples according to the method of Gleich and others by adding the same volume of physiological saline, mixing it for one minute by a vortex mixer and centrifuging at 40000 Xg, 4 C, for 30 minutes. We found that the measurement of ECP is not inhibited by materials in sputum supernatants through a dilution test and a recovery test. We also found that the followings did not influence the measurement of ECP; the times of dilution, the duration from collection to handing of sputum, the solution added to sputum, and the gravity of centrifugation. In addition, it was suggested that only ECP released into sputum is measurable from the results of an electron microscopic study and the measurement of ECP after centrifugation following the addition of white blood cells to sputum.

Asthma↗
PubMed1996

Haemoglobin adducts as biomarkers of occupational exposure to 1,3-butadiene.

Adducts of 1,2-epoxy-3-butene (EB) with haemoglobin were monitored in 17 workers from the 1,3-butadiene (BD) production unit of a petrochemical plant and in nine referents employed at the same factory but not exposed to BD. The air concentrations of BD were determined using stationary and personal monitoring. The ambient level of exposure of the plant workers handling butadiene containers (sampling and voiding) was on average 11.2 +/- 18.6 (mean +/- SD) mg/m3. Maintenance and laboratory workers were exposed to levels < or = 1.2 mg/m3. The particular haemoglobin adduct measured was 2-hydroxy-3-butenylvaline, formed by reaction of N-terminal valine with carbon 1 in EB. The adduct levels were increased (0.16 +/- 0.099 pmol/g; n = 10) in plant workers compared with the levels in maintenance and laboratory workers and controls (approximately 0.05 pmol/g; seven laboratory workers and nine controls evaluated). Thus, the method used for adduct determination--derivatization of 200-300 mg globin samples with penta-fluorophenyl isothiocyanate according to the N-alkyl Edman method and detection of the thiohydantoin derivatives by tandem mass spectrometry--is sufficiently sensitive to allow monitoring of exposure to BD down to the p.p.m. level.

Amino Acid Sequence↗
PubMed2005

Reproducibility and accuracy of measurements of free and total prostate-specific antigen in serum vs plasma after long-term storage at -20 degrees C.

BACKGROUND: Long-term frozen storage may alter the results of prostate-specific antigen (PSA) measurements, mainly because of degradation of free PSA (fPSA) in vitro. We compared the effects of long-term storage on fPSA, total PSA (tPSA), and complexed PSA (cPSA) in serum vs EDTA-plasma samples. METHODS: We measured fPSA and tPSA concentrations in matched pairs of archival serum and EDTA-plasma samples (stored frozen at -20 degrees C for 20 years) from a large population-based cohort in Malmö, Sweden. We also compared concentrations in age-matched men with those in samples not subjected to long-term storage, obtained from participants in a population-based study of prostate cancer screening in Göteborg, Sweden. These contemporary samples were handled according to standardized preanalytical and analytical protocols aimed at minimizing in vitro degradation. tPSA and fPSA measurements were performed with a commercial assay (Prostatus Dual Assay; Perkin-Elmer Life Sciences). RESULTS: Concentrations of tPSA and fPSA and calculated cPSA (tPSA - fPSA) in archival plasma were not significantly different from those in contemporary serum from age-matched men. In archival serum, however, random variability of fPSA was higher vs plasma than in contemporary samples, whereas systematic error of fPSA analyses was similarly small in archival and contemporary serum and plasma. CONCLUSIONS: Concentrations of tPSA and calculated cPSA were highly stable in plasma and serum samples subjected to long-term storage at -20 degrees C. Greater random variability, rather than a systematic decrease, may explain differences in fPSA analyses observed in archival serum.

Aged↗
PubMed1988

[Mail handling: a possible cause of error in the non-selective screening for congenital hypothyroidism].

Remittance by mail of blood samples and subsequent time of permanency in mail boxes are not supposed to be best thermic conditions for dried blood samples in paper used for neonatal screening. With the idea of checking if temperatures variations can altered or not the content of TSH and T4, some samples of known concentrations of TSH and T4 has been heated to different temperatures between less than -40 degrees C and 100 degrees C during weekly periods. At such temperatures between less than -40 degrees C and 25 degrees C significant hormones losses are not observed. Nevertheless under higher temperatures the percentages of losses increases. Specifically between 37 degrees C and 60 degrees C an hormonal loss of approximately 36% is observed. T4 had also suffered losses of concentration in relation with temperature. These variations make indispensable the change of the way of managing samples that could had suffered modifications with temperatures, and that have been sent by mail. Therefore, considering that about a 40% loss can exist, limit of 40 mu UI/ml should be modified about 25 mu UI/ml.

Blood Specimen Collection↗
PubMed1987

Free and total insulin as determined after precipitation with polyethylene glycol: analytical characteristics and effects of sample handling and storage.

We evaluated results of radioimmunoassays of free and total insulin after precipitation of endogenous antibodies with polyethylene glycol (PEG), and we investigated the influence of collection time, temperature, and storage in heparin- cr EDTA-treated plasma or serum on results for free insulin. Analytical recovery of free insulin was 99.3%, of total insulin 96.4%. For free insulin, assay precision (CV) was 4.0-13.0% (intra-assay) and 7.8-10.7% (inter-assay); for total insulin, 3.6-9.5% and 6.6-11.7%, respectively. Free insulin decreased in plasma (p less than 0.05) and serum (p less than 0.01) at room temperature after 3 h and in promptly analyzed serum (p less than 0.01). Storage of samples at -20 degrees C increased the concentration of free insulin in plasma (p less than 0.025) and serum (p less than 0.005), whereas the free insulin content of supernates after PEG precipitation was stable, except for a slight decrease in serum samples (p less than 0.02). We conclude that, for radioimmunoassay of free and total insulin, plasma should be used, treated with PEG without delay; supernates then are analytically stable for as long as 26 weeks at -20 degrees C.

Antibodies↗
PubMed2006

Influence of storage temperature on the stability of HIV-1 RNA and HSV-2 DNA in cervicovaginal secretions collected by vaginal washing.

Variability in the handling of samples of genital secretions prior to quantitation of HIV-1 RNA and HSV DNA may profoundly affect both the detection and quantitation of these nucleic acids. Over 144 h, we evaluated, the influence of storage temperature (4 degrees C, 20 degrees C, 30 degrees C) on the quantity of HIV-1 RNA and HSV-2 DNA in HIV and HSV negative cervicovaginal lavage pools spiked with known amounts of HIV-1 and HSV-2 and in HIV-1 and HSV-2 co-infected cervicovaginal lavage pools. The level of viral nucleic acids remained stable at 4 degrees C for 24h but decreased significantly when cervicovaginal lavages were stored at 20 degrees C and 30 degrees C, demonstrating that, cervicovaginal lavages to be quantified for viral RNA or DNA require, at minimum, immediate storage at 4 degrees C.

Cervix Uteri↗
PubMed2006

Versatile gas/particle ion chromatograph.

A new, compact gas/particle ion chromatograph has been developed for measuring ionic constituents in PM2.5 (particulate matter of aerodynamic diameter < or = 2.5 microm) and water-soluble ionogenic gases. The instrument has separate sampling channels for gases and particles. In one, a membrane denuder collects soluble gases for preconcentration and analysis. In the other, a cyclone removes larger particles, a membrane denuder removes soluble gases, and a continuously wetted hydrophilic filter collects particles. A single, multiport, syringe pump handles liquid transport, and one conductivity detector measures anions and ammonium for both channels. Electrodialytically generated gradient hydroxide eluent permits 20 min chromatographic runs. Gas/particle samples are each collected for 40 min, butthe sampling intervals are staggered by 20 min. Liquid samples from the gas denuder and particle collector are aspirated and preconcentrated on sequential cation and anion concentrators and transferred respectively to an ammonia transfer device and an anion separation column. The flow configuration results in an ammonium peak before anion peaks in the chromatogram. The system measures ammonia, organic acids (such as acetic, formic, and oxalic acids), HCl, HONO, SO2, HNO3, and the corresponding ions in the aerosol phase. Low ng/m3 to sub-ng/m3 limits of detection (LODs) are attained for most common gases and particulate constituents, the LODs for gaseous SO2 to NH3 range, for example, from sub parts per trillion by volume (sub-pptv) to approximately 5 pptv.

Aerosols↗
PubMed2004

Automated 96-well liquid-liquid back extraction liquid chromatography-tandem mass spectrometry method for the determination of ABT-202 in human plasma.

A high-throughput bioanalytical method using automated sample transferring, automated liquid-liquid back extraction and liquid chromatography-tandem mass spectrometry was developed in a GLP regulated environment for the determination of ABT-202 in human plasma. Samples of 0.30 ml were transferred into 96-well plate using an automatic liquid handler. Automated liquid-liquid extraction (LLE) was carried out on a 96-channel programmable liquid handling workstation using methyl tert-butyl ether as the extraction solvent. A dual-HPLC with single mass spectrometer configuration was utilized to provide a reliable and routine means to increase sample throughput. The standard curve range was 0.38-95.02 ng/ml. There was no interference from endogenous components in the blank plasma tested. The accuracy (% bias) at the lower limit of quantitation (LLOQ) was 7.7% and the precision (% CV) for samples at the LLOQ was 4.7%. The inter-day % CV and % bias of the quality control samples were < or = 6.8 and < or = 7.6%, respectively. Coefficients of determination, a measure of linearity, ranged from 0.994 to 0.997. The method was accurate and reproducible and was successfully applied to generate plasma concentration-time profiles for human subjects after low oral doses of the compound.

Administration, Oral↗
PubMed2003

Evaluation of a PCR test for the diagnosis of Tritrichomonas foetus infection in bulls: effects of sample collection method, storage and transport medium on the test.

The objectives of this study were to determine the detection limit of a PCR test for Tritrichomonas foetus, to investigate the effect of sampling method, guanidinium thiocyanate (GuSCN), and sample storage, and to confirm the accuracy of the test on field samples. Serial 10-fold dilutions of culture material were used to determine the detection limit. For the sample handling trial, five positive bulls were sampled by sheath washing and scraping on six occasions over a period of 18 days (n=29 samples) and eight control bulls were sampled three, four or six times (n=28 samples). Samples were cultured, while portions with and without GuSCN were subjected to DNA extraction within 6h, after 30 h and after 5 days at 4 degrees C. PCR and agarose gel electrophoresis was performed. A two-tailed chi-square test was used to test for differences between treatments. The PCR assay showed a specificity of 98%. Its sensitivity declined with storage time, from 90% at 6h to 31% at 5 days. Sampling method and GuSCN had no effect on test sensitivity. The detection limit of the assay was 100 organisms. Parallel testing of 193 field samples gave complete agreement between culture and PCR results.

Animals↗
PubMed2005

Effects of intraoperative fluid infusions, sample storage time, and sample handling on unbound propofol assay in human blood plasma.

Free drug assay in physiological fluids is getting more and more attention nowadays. The principal reason is the fact that the unbound drug form is responsible for the therapeutic or toxic effects of its application. Unbound drug concentration significantly depends on the extent of its binding by plasma. This article describes the influence of different factors on unbound propofol concentration. These factors are presence of infusion fluids in blood, type of anticoagulant, sample storage time and plasma freezing. The following conclusions result from the experiments carried out: 1. The lowest free drug fractions are observed in samples containing carbohydrate infusion fluids. The free drug percentage is virtually independent of its total concentration in the range of clinically relevant concentrations. 2. There is no evident anticoagulant influence (heparin, citrate, EDTA and oxalate) on free propofol level in plasma samples. 3. Longer storage of plasma at 4 degrees C causes a slight rise of free propofol concentration in heparinised plasma and no evident changes in plasma containing citrate. 4. Plasma freezing induces the increase of free drug concentration both for citrate and heparin. These findings are valuable both for clinicists and pharmacologists, and important for chemical analysts.

Drug Storage↗
PubMedDate not supplied

[Pharmacy Department standard procedures for clinical trial development].

INTRODUCTION: Regulations on good clinical practice in the execution of clinical trials require the establishment and compliance of normalised working procedures by the hospital Pharmacy Department. These procedures must indicate precisely each one of the processes involved in their development, e.g. the reception, custody, storage, preparation and dispensing of research samples. OBJECTIVE: The aim of the present study is to analyse the information included in clinical trial protocols involved in the circulation of drug samples during the research stage and, based on the results obtained, to develop normalised working procedures for our hospital Pharmacy Department in order to carry out these clinical trials. MATERIAL AND METHODS: Eleven items were defined in order to describe key processes involved in identification, storage, receipt, preparation, dispensation, administration, maintenance and return of research samples, as well as those related to the beginning and end or closing of a clinical trial. Information included in 39 clinical trial protocols that had been sent to the Hospital Ethics Committee for evaluation between January and August of 2002 and was analysed in terms of the items previously defined. RESULTS: 46% of the protocols included data from the monitor or person representing the sponsor. 64% adequately described conservation and storage conditions of the research samples; but in 39% of the studied cases external labelling descriptions did not comply with legislation. Only 15% of the cases included clear instructions on research sample reception and its acknowledgement. 25.6% reported correct handling and/or preparation instructions for research samples with parenteral administration. Sample dispensation procedures were correctly described in no more than 5.1% of the protocols analyzed. 41% included information for patients, relatives or nurses on administration and handling of research samples, but none of them included specific information pamphlets or three-page leaflets. Procedures for either returning research samples to the sponsor or for their maintenance and replacement were not specified in the 71.8 and 100% of the cases, respectively. Likewise, trial ending or closing dates as well as procedures that must be followed by the Hospital Pharmacy Department during these processes were not stated in 89.7% of the protocols. Finally, only 30.8% of the cases explicitly cite the hospital Pharmacy Department. CONCLUSION: These results support and justify the need to improve clinical trial protocols in order for them to be performed in a secure, effective and efficient way. Besides, they have been the starting point to devise the normalised working procedures of the hospital Pharmacy Department for the development of the clinical trials.

Clinical Protocols↗
PubMedDate not supplied

The effect of refrigeration and mixing on detection of endotoxin in parenteral drugs using the Limulus Amebocyte Lysate (LAL) test.

Prior to testing for the presence of bacterial endotoxin, parenteral products are handled and stored in a variety of ways. Two incidents, detected by the U.S. Food and Drug Administration, revealed that differences in product handling and storage may have played a role in causing analytical discrepancies in the testing of identical samples. The testing procedure was the USP Bacterial Endotoxin test using Limulus Amebocyte Lysate (LAL) reagent. Consequently, an evaluation was made at the two principal factors that contributed to the suspected analytical anomaly. The factors were sample storage and the degree of agitation prior to sample analysis. Additional variables such as bacterial growth medium and adsorption potential of endotoxin by rubber stoppers were also evaluated. It was found that neither the medium employed to grow the E. coli endotoxin nor the storage temperature of the spiked solutions were problematic. However, it was shown that 20-40% of the spiked endotoxin was lost due to non-agitation of solution in vials in which the solution was in contact with the rubber stoppers. A suggested remedy for this problem is to store intact product containers in an upright position and to establish a uniform mixing procedure prior to endotoxin assay.

Endotoxins↗
PubMed2005

NanoStore: a concept for logistical improvements of compound handling in high-throughput screening.

Small molecule screening, the systematic encounter of biology space with chemical space, has provoked the emergence of a whole industry that recreates itself by constant iterative improvements to this process. The authors describe an approach to tackle the problem for one of the most time-consuming steps in the execution of a screening campaign, namely, the reformatting of high-throughput screening test compounds from master plates to daughter assay plates used in the execution of the screen. Through an engineered storage procedure, they prepare plates ahead of the screening process with the respective compounds in a ready-to-use format. They show the biological inertness of the method and how it facilitates efficient recovery of compound activity. This uncoupling of normally interconnected processes provides time and compound savings, avoids repeated freeze-thaw cycles of compound solutions, and removes the problems associated with the DMSO sensitivity of certain assays types.

Automation↗
PubMed1987

Sample handling techniques when analyzing regulatory peptides.

Collection of blood samples in prechilled heparinized tubes, rapid cooling and centrifugation at 4 degrees C were found to be more important than the enzyme inhibitors aprotinin and EDTA in preserving immunoreactive neuropeptide Y. Nine months after storage of plasma in the frozen state at -20 degrees C or -80 degrees C the recovery of NPY was about 50% of the recovery at immediate analysis. Synthetic substance P added to guinea pig plasma at 37 degrees C disappeared almost entirely within 30 seconds as measured by radioimmunoassay while the concentrations of neurokinin A and neuropeptide K decreased only to a minor extent during a 20 min observation period. The total concentration of immunoreactive substance P and neurokinin A in boiled aqueous and acetic acid extracts of rat dorsal spinal cord was on the other hand stable for 72 h at 4 degrees C, 24 h at room temperature and after freezing and thawing three times. However, chromatographic analysis indicated that the immunoreactivity became increasingly more heterogenous in the samples particularily at room temperature. Acid ethanol and Sep Pak extraction of plasma samples resulted in almost 90% recovery of neuropeptide Y, neuropeptide K and calcitonin gene-related peptide while removing crossreacting substances with high molecular weight.

Animals↗