Recent developments in the use of isotope ratio mass spectrometry in sports drug testing.

According to the annual report of the World Anti-Doping Agency, steroids are the most frequently detected class of doping agents. Detecting the misuse of endogenously occurring steroids, i.e. steroids such as testosterone that are produced naturally by humans, is one of the most challenging issues in doping control analysis. The established thresholds for urinary concentrations or concentration ratios such as the testosterone/epitestosterone quotient are sometimes inconclusive owing to the large biological variation in these parameters.For more than 15 years, doping control laboratories focused on the carbon isotope ratios of endogenous steroids to distinguish between naturally elevated steroid profile parameters and illicit administration of steroids. A variety of different methods has been developed throughout the last decade and the number of different steroids under investigation by isotope ratio mass spectrometry has recently grown considerably. Besides norandrosterone, boldenone was found to occur endogenously in rare cases and the misuse of corticosteroids or epitestosterone can now be detected with the aid of carbon isotope ratios as well. In addition, steroids excreted as sulfoconjugates were investigated, and the first results regarding hydrogen isotope ratios recently became available.All of these will be presented in detail within this review together with some considerations on validation issues and on identification of parameters influencing steroidal isotope ratios in urine.

Multicentre validation study of nucleic acids extraction from FFPE tissues.

In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp-250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.

Spare non-occupational HIV post-exposure prophylaxis by active contacting and testing of the source person.

OBJECTIVE: HIV-1 post-exposure prophylaxis (PEP) is frequently prescribed after exposure to source persons with an undetermined HIV serostatus. To reduce unnecessary use of PEP, we implemented a policy including active contacting of source persons and the availability of free, anonymous HIV testing ('PEP policy'). METHODS: All consultations for potential non-occupational HIV exposures i.e. outside the medical environment) were prospectively recorded. The impact of the PEP policy on PEP prescription and costs was analysed and modelled. RESULTS: Among 146 putative exposures, 47 involved a source person already known to be HIV positive and 23 had no indication for PEP. The remaining 76 exposures involved a source person of unknown HIV serostatus. Of 33 (43.4%) exposures for which the source person could be contacted and tested, PEP was avoided in 24 (72.7%), initiated and discontinued in seven (21.2%), and prescribed and completed in two (6.1%). In contrast, of 43 (56.6%) exposures for which the source person could not be tested, PEP was prescribed in 35 (81.4%), P < 0.001. Upon modelling, the PEP policy allowed a 31% reduction of cost for management of exposures to source persons of unknown HIV serostatus. The policy was cost-saving for HIV prevalence of up to 70% in the source population. The availability of all the source persons for testing would have reduced cost by 64%. CONCLUSION: In the management of non-occupational HIV exposures, active contacting and free, anonymous testing of source persons proved feasible. This policy resulted in a decrease in prescription of PEP, proved to be cost-saving, and presumably helped to avoid unnecessary toxicity and psychological stress.

Predictive ability of severe rainfall events over Catalonia for the year 2008

This paper analyses the predictive ability of quantitative precipitation forecasts (QPF) and the so-called "poor-man" rainfall probabilistic forecasts (RPF). With this aim, the full set of warnings issued by the Meteorological Service of Catalonia (SMC) for potentially-dangerous events due to severe precipitation has been analysed for the year 2008. For each of the 37 warnings, the QPFs obtained from the limited-area model MM5 have been verified against hourly precipitation data provided by the rain gauge network covering Catalonia (NE of Spain), managed by SMC. For a group of five selected case studies, a QPF comparison has been undertaken between the MM5 and COSMO-I7 limited-area models. Although MM5's predictive ability has been examined for these five cases by making use of satellite data, this paper only shows in detail the heavy precipitation event on the 9¿10 May 2008. Finally, the "poor-man" rainfall probabilistic forecasts (RPF) issued by SMC at regional scale have also been tested against hourly precipitation observations. Verification results show that for long events (>24 h) MM5 tends to overestimate total precipitation, whereas for short events (¿24 h) the model tends instead to underestimate precipitation. The analysis of the five case studies concludes that most of MM5's QPF errors are mainly triggered by very poor representation of some of its cloud microphysical species, particularly the cloud liquid water and, to a lesser degree, the water vapor. The models' performance comparison demonstrates that MM5 and COSMO-I7 are on the same level of QPF skill, at least for the intense-rainfall events dealt with in the five case studies, whilst the warnings based on RPF issued by SMC have proven fairly correct when tested against hourly observed precipitation for 6-h intervals and at a small region scale. Throughout this study, we have only dealt with (SMC-issued) warning episodes in order to analyse deterministic (MM5 and COSMO-I7) and probabilistic (SMC) rainfall forecasts; therefore we have not taken into account those episodes that might (or might not) have been missed by the official SMC warnings. Therefore, whenever we talk about "misses", it is always in relation to the deterministic LAMs' QPFs.

Soluble MHC-peptide complexes containing long rigid linkers abolish CTL-mediated cytotoxicity.

Soluble MHC-peptide (pMHC) complexes induce intracellular calcium mobilization, diverse phosphorylation events, and death of CD8+ CTL, given that they are at least dimeric and co-engage CD8. By testing dimeric, tetrameric, and octameric pMHC complexes containing spacers of different lengths, we show that their ability to activate CTL decreases as the distance between their subunit MHC complexes increases. Remarkably, pMHC complexes containing long rigid polyproline spacers (> or =80 A) inhibit target cell killing by cloned S14 CTL in a dose- and valence-dependent manner. Long octameric pMHC complexes abolished target cell lysis, even very strong lysis, at nanomolar concentrations. By contrast, an altered peptide ligand antagonist was only weakly inhibitory and only at high concentrations. Long D(b)-gp33 complexes strongly and specifically inhibited the D(b)-restricted lymphocytic choriomeningitis virus CTL response in vitro and in vivo. We show that complications related to transfer of peptide from soluble to cell-associated MHC molecules can be circumvented by using covalent pMHC complexes. Long pMHC complexes efficiently inhibited CTL target cell conjugate formation by interfering with TCR-mediated activation of LFA-1. Such reagents provide a new and powerful means to inhibit Ag-specific CTL responses and hence should be useful to blunt autoimmune disorders such as diabetes type I.

More on Testing for Validity Instead of Looking for It

Using Monte Carlo simulations and reanalyzing the data of a validation study of the AEIM emotional intelligence test, we demonstrated that an atheoretical approach and the use of weak statistical procedures can result in biased validity estimates. These procedures included stepwise regression-and the general case of failing to include important theoretical controls-extreme scores analysis, and ignoring heteroscedasticity as well as measurement error. The authors of the AEIM test responded by offering more complete information about their analyses, allowing us to further examine the perils of ignoring theory and correct statistical procedures. In this paper we show with extended analyses that the AEIM test is invalid.

MALDI-TOF MS-based drug susceptibility testing of pathogens: The example of Candida albicans and fluconazole.

MALDI-TOF MS can be used for the identification of microorganism species. We have extended its application to a novel assay of Candida albicans susceptibility to fluconazole, based on monitoring modifications of the proteome of yeast cells grown in the presence of varying drug concentrations. The method was accurate, and reliable, and showed full agreement with the Clinical Laboratory Standards Institute's reference method. This proof-of-concept demonstration highlights the potential for this approach to test other pathogens.