Workshop report on the extraction of foetal DNA from maternal plasma

Objective Cell free foetal DNA (cff DNA) extracted from maternal plasma is now recognized as a potential source for prenatal diagnosis but the methodology is currently not well standardized. To evaluate different manual and automated DNA extraction methods with a view to developing standards, an International Workshop was performed. Methods Three plasma pools from RhD-negative pregnant women, a DNA standard, real-time-PCR protocol, primers and probes for RHD were sent to 12 laboratories and also to one company (Qiagen, Hilden, Germany). In pre-tests, pool 3 showed a low cff DNA concentration, pool 1 showed a higher concentration and pool 2 an intermediate concentration. Results The QIAamp DSP Virus Kit, the High Pure PCR Template Preparation Kit, an in-house protocol using the QIAamp DNA Blood Mini Kit, the CST genomic DNA purification kit, the Magna Pure LC, the MDx, the M48, the EZ1 and an in-house protocol using magnetic beads for manual and automated extraction were the methods that were able to reliably detect foetal RHD. The best results were obtained with the QIAamp DSP Virus Kit. The QIAamp DNA Blood Mini Kit showed very comparable results in laboratories that followed the manufacturer's protocol and started with ≥ 500 µL plasma. One participant using the QIAamp DNA Blood Midi Kit failed to detect reliably RHD in pool 3. Conclusions This workshop initiated a standardization process for extraction of cff DNA in maternal plasma. The highest yield was obtained by the QIAamp DSP Virus Kit, a result that will be evaluated in more detail in future studies. Copyright © 2007 John Wiley & Sons, Ltd.     

Evaluation of a simultaneous sampling method of PAHs, PCDD/Fs and dl-PCBs in ambient air

The Stockholm Convention on Persistent Organic Pollutants (POPs) was signed in May 2001 by 127 countries. Currently, 12 substances are regulated by the convention, and the work on finding new candidate chemicals to the convention has started. Among these 12 substances, dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) are the objects of this study. There are no European standard methods for the simultaneous determination of these microorganic compounds, together with polycyclic aromatic hydrocarbons (PAHs) in ambient air--they must be referred to three different methods developed by the U.S. Environmental Protection Agency (EPA). The quali-quantitative analysis of these microorganic pollutants is an important challenge due to the low concentrations at which they may be present. In this study, the development of a simplified, alternative, fast and affordable sampling method for the determination of PAHs, PCDDs, PCDFs and PCBs in ambient air was performed. The sampling time was extended from 24 h to 7 d in order to enrich the sample, to fall within the instrumental limits of detection and to reduce the number of samples to be processed and, therefore, errors that may arise. First of all, experiments with labelled standards were conducted in the research area of Montelibretti (rural station, which is sited about 20 km northeast of Rome), with the purpose of optimizing sampling efficiency. Finally, the method was applied to the analysis of these compounds in the air of a suburban area with small industrial plants in order to evaluate the feasibility of the proposed sampling method system, by comparing concentrations of native compounds acquired during simultaneous daily and weekly sampling.     

Characterization of post-consumer polyolefin wastes by hyperspectral imaging for quality control in recycling processes

In this paper new analytical inspection strategies, based on hyperspectral imaging (HSI) in the VIS–NIR and NIR wavelength ranges (400–1000 and 1000–1700 nm, respectively), have been investigated and set up in order to define quality control logics that could be applied at industrial plant level for polyolefins recycling. The research was developed inside the European FP7 Project W2Plastics “Magnetic Sorting and Ultrasound Sensor Technologies for Production of High Purity Secondary Polyolefins from Waste”. The main aim of the project is the separation of pure polyethylene and polypropylene adopting an innovative process, the magnetic density separation (MDS). Spectra of plastic particles and contaminants resulting from post-consumer complex wastes and of virgin polyolefins have been acquired by HSI and by Raman spectroscopy. The classification results obtained applying principal component analysis (PCA) on HSI data have been compared with those obtained by Raman spectroscopy, in order to validate the proposed innovative methodology. Results showed that HSI sensing techniques allow to identify both polyolefins and contaminants. Results also demonstrated that HSI has a great potentiality as a tool for quality control of feed (identification of contaminants in the plastic waste) and of the two different pure polypropylene and polyethylene flow streams resulting from the MDS-based recycling process.     

Evaluation of an Exposure Assay to Measure Uptake of Sediment Pah by Fish

The ecological risks of polynuclear aromatic hydrocarbons (PAH) in aquatic sediments will vary with both toxicity and bioavailability to aquatic biota. While there are standardized protocols to test the acute toxicity of sediment-borne compounds to aquatic invertebrates, there are none for assessing bioavailability to fish. We found that sediment-borne PAH caused an exposure-dependent induction of cytochrome P450 (CYP1A) enzymes in rainbow trout (Oncorhynchus mykiss) fingerlings exposed in semi-static 96 h bioassays, as shown by increased activity of ethoxyresorufin-o-deethylase (EROD). Assuming that PAH are taken up by trout due to partitioning from organic and inorganic constituents of sediments, we tested the effect of different test variables on bioaccumulation using induction as an index of exposure. EROD activity increased with exposure of fish to increasing volumes of sediments containing PAH, i.e., with increasing ratios of sediment to water. Uptake of single compounds from sandy sediments did not differ from uptake from clay or low organic (7% LOI — loss on ignition) sediments, but decreased when organic content was very high (58% LOI). Maximum induction was observed within 24~h of exposure and at 7.5^C relative to 15, 22, or 28^C. Storage and handling techniques had minor effects on sediment EROD induction potency. Absolute EROD activity was greater in white sucker (Catostomus commersoni) a benthic species, than in trout, a pelagic species. However, when basal (negative control) activity was accounted for, there was no difference in response between the species. Together, these experiments provide a basis for developing a standard protocol to test the bioavailability to fish of sediment-borne PAH.     

Contamination Levels and Preliminary Assessment of the Technical Feasibility of Employing Natural Attenuation in 5 Priority Areas of Presidente Bernardes Refinery in Cubatão, São Paulo, Brazil

Five priority areas of potential impact by contaminants (API) were investigated at the Presidente Bernardes Refinery in Cubatão, São Paulo, Brazil with the following aims: (i) to identify both organic and inorganic contaminants present in soil and groundwater; (ii) to define the environmental conditions relevant for microbial activity at the site and (iii) to evaluate the feasibility of employing natural attenuation for treatment of the hydrocarbon contamination. One area (API 1) was an uncontrolled landfill, where waste materials from the refinery were deposited between 1954 and 1986, and four areas (API 4, 5, 7 and 11) were located in the operational section of the refinery. Soil contamination by regulated BTEX compounds (benzene, toluene, total xylenes) was restricted to two samples from API 1. Nonregulated ethylbenzene was detected in one soil sample from API 4, one from API 5 and two from API 1. No soil contained regulated PAH above threshold levels. Several nonregulated PAHs were found in 6 soil samples from API 1, 3 soil samples from API 4 and 1 soil sample from API 5. Site soils contained very high aluminium concentrations, but metal contamination was restricted to one soil sample from API 1, which contained nickel above threshold limits. BTEX contamination of groundwater was due mostly to benzene. Of the 17 PAH molecules tested, only naphthalene and 2-methylnaphthalene occurred in groundwater. The sum of total BTEX and total PAH exceeded 200 μg/L in only a few monitoring wells in API 4, 5 and 11 and was always below 2.640 μg/L. Be, Cd, Cr, Cu, Hg, Ni, Se, Ag, Tl and Zn were not detected in groundwater, which was contaminated in a few locations by aluminium (mostly below 1 mg/L), lead (<0.066 mg/L) and arsenic (<0.056 mg/L). S, K, Ca, Mg and Fe were present in groundwater in excess of physiological requirements for microbial growth, but low concentrations of N and P could become growth limiting. However, BTEX were efficiently degraded in saturated and unsaturated zone microcosms and nutrient amendments did not stimulate biodegradation rates measurably. The inorganic carbon pool in groundwater was up to one order of magnitude larger than the organic carbon pool. Total inorganic carbon (TIC) in API groundwater exceeded TIC of clean groundwater by factors of 2 (API 4), 6 (API 5, 7 and 11) or 10 (API 1). Most of the inorganic carbon incorporated into groundwater beneath the refinery originated from biodegradation in the unsaturated soil, which contained a microbiota (10⁶ cells/g on average) capable of growth with most of the pure (benzene, toluene, ethylbenzene and xylene) and mixed hydrocarbons tested (diesel oil, gasoline, naphtha, condensate, aromatic residue and fuel oil). A viscous hydrocarbon paste uncovered in API 1 was insoluble in water but dissolved in dichloromethane. Many organic components of this paste were biodegradable as evidenced by weight reduction of the hydrocarbon paste and by the growth of suspended and attached biomass in saturated zone microcosms, where the paste was the only carbon source. This study indicates that monitored natural attenuation may be a technically feasible and efficient means for plume control in API 1, 4 and 5, provided the plumes in API 4 and 5 are not expanding. This technique is not suitable for contaminant reduction in API 11.     

Prenatal testing for imprinting disorders: A laboratory perspective

Imprinting Disorders (ImpDis) are a group of congenital syndromes associated with up to four different types of molecular disturbances affecting the monoallelic and parent-of-origin specific expression of genomically imprinted genes. Though each ImpDis is characterized by aberrations at a distinct genetic site and a specific set of postnatal clinical signs, there is a broad overlap between several of them. In particular, the prenatal features of ImpDis are non-specific. Therefore, the decision on the appropriate molecular testing strategy is difficult. A further molecular characteristic of ImpDis is (epi)genetic mosaicism, which makes prenatal testing for ImpDis challenging. Accordingly, sampling and diagnostic workup has to consider the methodological limitations. Furthermore, the prediction of the clinical outcome of a pregnancy can be difficult. False-negative results can occur, and therefore fetal imaging should be the diagnostic tool on which decisions on the management of the pregnancy should be based. In summary, the decision for molecular prenatal testing for ImpDis should be based on close exchanges between clinicians, geneticists, and the families before the initiation of the test. These discussions should weigh the chances and challenges of the prenatal test, with focus on the need of the family.