A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Introduction to the DAPPLE Air Pollution Project.

The Dispersion of Air Pollution and its Penetration into the Local Environment (DAPPLE) project brings together a multidisciplinary research group that is undertaking field measurements, wind tunnel modelling and computer simulations in order to provide better understanding of the physical processes affecting street and neighbourhood-scale flow of air, traffic and people, and their corresponding interactions with the dispersion of pollutants at street canyon intersections. The street canyon intersection is of interest as it provides the basic case study to demonstrate most of the factors that will apply in a wide range of urban situations. The aims of this paper are to introduce the background of the DAPPLE project, the study design and methodology for data collection, some preliminary results from the first field campaign in central London (28 April-24 May 2003) and the future for this work. Updated information and contact details are available on the web site at http://www.dapple.org.uk.

Use of capillary gas chromatography with negative ion-chemical ionization mass spectrometry for the determination of perfluorocarbon tracers in the atmosphere.

A sensitive and selective technique for the quantitative measurement of atmospheric perfluorocarbon trace species at the sub part per quadrillion (10(-15)) levels is presented. The method utilizes advances in adsorbent enrichment techniques coupled with benchtop capillary gas chromatography and negative ion-chemical ionization mass spectrometry. The development and enhancement of sampling technology for tracer experiments is described, and the results from background measurements and a preliminary field experiment are presented. The overall precision of the analytical method with respect to the preferred tracer for these atmospheric transport studies, perfluoromethylcyclohexane, was +/-1.7%. The background concentrations of perfluorodimethylcyclobutane, perfluoromethylcyclopentane, and perfluoromethylcyclohexane at a remote coastal location (Mace Head, Ireland, 53 degrees N, 10 degrees W) were found to be 2.5 (+/-0.4), 6.8 (+/-1.0), and 5.2 fL L(-1) (+/-1.3), respectively. Background concentrations within an urban conurbation (Bristol, U.K.) were slightly greater at 3.0 (+/-1.5), 8.1 (+/-1.8), and 6.3 fL L(-1) (+/-1.1), respectively.

An automated system for near-real-time monitoring of trace atmospheric halocarbons.

A new gas chromatographic method developed to quantitatively determine important atmospheric halocarbons is described. Target compounds include replacement CFCs, chlorinated solvents, and biosynthesized (naturally produced) organohalogens, all trace gases in the atmosphere at concentrations ranging from 0.1 to 600 pptv (where pptv = 1 part in 10(-)(12) by volume). A combination of ultralow concentrations and relatively small electron attachment cross sections renders these compounds very difficult to routinely measure in the background air typical of remote atmospheric monitoring stations. Detection is achieved by preconcentration of a 200-mL air sample using an adsorbent-filled microtrap and enhancement of electron capture detector response by oxygen doping one of two detectors connected in series. Oxygen doping specifically targets halocarbons with relatively poor electron attachment rate coefficients. The work described here details construction of a novel analytical system, laboratory trials, and optimization followed by an extended field campaign at a remote atmospheric monitoring station, Mace Head, Ireland. A calibration standard or ambient air sample was acquired every hour using a cyclic, automated procedure without employing cryogenic preconcentration or refocusing. Overall precision of the analytical method for the target compounds is between 0.3 and 1.5%.

Chemometric studies of the effect of toxic metals on plants: the use of response surface methodology to investigate the influence of Tl, Cd and Ag on the growth of cabbage seedlings.

The growth of cabbage seedlings at five concentrations of Cd, Tl and Ag was monitored by measuring their wet weights, root and shoot lengths after four week's growth in a controlled environment. Toxicity curves were constructed using a three factor central composite experimental design. The interaction and competition between any two metals were studied by displaying response surfaces. Ag was observed to be the most toxic, while Tl and Cd, although toxic, exhibited fairly similar effects. Analysis of variance was used to test for the significance of each of the computed parameters.

A rapid chemiluminescent enzyme-linked immunosorbent assay for cytomegalovirus immunoglobulin G antibodies using instant photographic film.

A rapid and convenient chemiluminescent enzyme-linked immunosorbent assay (ELISA) for IgG antibodies to cytomegalovirus has been developed which uses low cost equipment. Assays were carried out on transparent microtitre plates and used an anti-human IgG horseradish peroxidase conjugate. Bound peroxidase was detected chemiluminescently using a p-iodophenol-luminol-peroxide reagent. Light emission from the wells of the microtitre plate was detected on instant photographic film (ASA 20,000) held in a specially designed shutter type camera. The semi-quantitative technique was tested in a routine laboratory for a period of 7 wk and the results obtained compared well (95.3% agreement) with those obtained by a conventional colorimetric ELISA using an alkaline phosphatase label.

Rapid determination of DDT and related compounds in soils via carbon skeleton gas chromatography-mass spectrometry.

The levels of DDT and related compounds in soil samples from an apple orchard have been determined. Extraction of residues was achieved by cyclic steam extraction and by conventional solvent extraction methods allowing comparison of the two methods. Determination of the organochlorine residues present in the extracts was performed by gas-liquid chromatography-electron-capture detection and by carbon skeleton gas chromatograph-mass spectrometry. Excellent agreement between the two determination techniques was achieved. Prior to application of p,p'-DDT, a value of approximately 0.4 ppm was obtained for Delta DDT in the soil. The major component of this total was DDE. After spraying the apple trees with technical grade, p,p'-DDT, Delta DDT for the soil rose to 0.6 ppm. This increase was due to translocation of p,p'-DDT from the trees.

Heavy metal complexation in polluted molluscs. III. Periwinkles (Littorina littorea), cockles (Cardium edule) and scallops (Chlamys opercularis).

The accumulation of cadmium, zinc and copper by environmentally contaminated periwinkles, cockles and scallops has been studied by gel permeation chromatography of water-soluble extracts of the shellfish. Three distinct low mol. wt. (less than 3000) zinc complexes were separated from periwinkle extracts. Copper and cadmium were present as a single low mol. wt. species which is apparently responsible for the elevated copper levels in Littorina littorea.

Heavy metal complexation in polluted molluscs II. Oysters (Ostrea edulis and Crassostrea gigas).

Cadmium, zinc and copper, accumulated from polluted habitats by the oysters Ostrea edulis and Crassostrea gagas, were studied. Three distinct low molecular weight (less than 3000 daltons) zinc complexes were separated from highly contaminated C. gigas, one of which may be a complex with the amino acid taurine. Only one of these zinc complexes was present in less contaminated specimens of O. edulis. On gel permeation chromatography of oyster extracts, copper was eluted together with amino acids (principally taurine) and the betaine homarine. No evidence of metallothionein type proteins was found.

Heavy metal complexation in polluted molluscs. I. Limpets (Patella vulgata and Patella intermedia).

The accumulation of cadmium, zinc and copper by the marine gastropod molluscs Patella vulgata and Patella intermedia has been studied by gel permeation chromatography of water-soluble extracts of environmentally contaminated shellfish. A major proportion of the water-soluble cadmium and copper in these molluscs is associated with a protein of molecular weight 10 800 daltons. Evidence is presented supporting the similarity of this protein with mammalian metallothionein. This protein contained only a small proportion of the zinc found in the samples.