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.

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.

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.