Results of An International Study of Quantitative BCR/ABL Assays Using Defined RNA Samples

Molecular monitoring of BCR/ABL transcripts by real time quantitative reverse transcription PCR (qRT-PCR) is an essential technique for clinical management of patients with BCR/ABL-positive CML and ALL. Though quantitative BCR/ABL assays are performed in hundreds of laboratories worldwide, results among these laboratories cannot be reliably compared due to heterogeneity in test methods, data analysis, reporting, and lack of quantitative standards. Recent efforts towards standardization have been limited in scope. Aliquots of RNA were sent to clinical test centers worldwide in order to evaluate methods and reporting for e1a2, b2a2, and b3a2 transcript levels using their own qRT-PCR assays. Total RNA was isolated from tissue culture cells that expressed each of the different BCR/ABL transcripts. Serial log dilutions were prepared, ranging from 100 to 10-5, in RNA isolated from HL60 cells. Laboratories performed 5 independent qRT-PCR reactions for each sample type at each dilution. In addition, 15 qRT-PCR reactions of the 10-3 b3a2 RNA dilution were run to assess reproducibility within and between laboratories. Participants were asked to run the samples following their standard protocols and to report cycle threshold (Ct), quantitative values for BCR/ABL and housekeeping genes, and ratios of BCR/ABL to housekeeping genes for each sample RNA. Thirty-seven (n=37) participants have submitted qRT-PCR results for analysis (36, 37, and 34 labs generated data for b2a2, b3a2, and e1a2, respectively). The limit of detection for this study was defined as the lowest dilution that a Ct value could be detected for all 5 replicates. For b2a2, 15, 16, 4, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. For b3a2, 20, 13, and 4 labs showed a limit of detection at the 10-5, 10-4, and 10-3 dilutions, respectively. For e1a2, 10, 21, 2, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. Log %BCR/ABL ratio values provided a method for comparing results between the different laboratories for each BCR/ABL dilution series. Linear regression analysis revealed concordance among the majority of participant data over the 10-1 to 10-4 dilutions. The overall slope values showed comparable results among the majority of b2a2 (mean=0.939; median=0.9627; range (0.399 - 1.1872)), b3a2 (mean=0.925; median=0.922; range (0.625 - 1.140)), and e1a2 (mean=0.897; median=0.909; range (0.5174 - 1.138)) laboratory results (Fig. 1-3)). Thirty-four (n=34) out of the 37 laboratories reported Ct values for all 15 replicates and only those with a complete data set were included in the inter-lab calculations. Eleven laboratories either did not report their copy number data or used other reporting units such as nanograms or cell numbers; therefore, only 26 laboratories were included in the overall analysis of copy numbers. The median copy number was 348.4, with a range from 15.6 to 547,000 copies (approximately a 4.5 log difference); the median intra-lab %CV was 19.2% with a range from 4.2% to 82.6%. While our international performance evaluation using serially diluted RNA samples has reinforced the fact that heterogeneity exists among clinical laboratories, it has also demonstrated that performance within a laboratory is overall very consistent. Accordingly, the availability of defined BCR/ABL RNAs may facilitate the validation of all phases of quantitative BCR/ABL analysis and may be extremely useful as a tool for monitoring assay performance. Ongoing analyses of these materials, along with the development of additional control materials, may solidify consensus around their application in routine laboratory testing and possible integration in worldwide efforts to standardize quantitative BCR/ABL testing.

Food habits and sport activity during adolescence: differences between athletic and non-athletic teenagers in Switzerland.

OBJECTIVE: To describe food habits and dietary intakes of athletic and non-athletic adolescents in Switzerland. SETTING: College, high schools and professional centers in the Swiss canton of Vaud. METHOD: A total of 3,540 subjects aged 9-19 y answered a self-reported anonymous questionnaire to assess lifestyles, physical plus sports activity and food habits. Within this sample, a subgroup of 246 subjects aged 11-15 also participated in an in-depth ancillary study including a 3 day dietary record completed by an interview with a dietician. RESULTS: More boys than girls reported engaging in regular sports activities (P<0.001). Adolescent food habits are quite traditional: up to 15 y, most of the respondents have a breakfast and eat at least two hot meals a day, the percentages decreasing thereafter. Snacking is widespread among adolescents (60-80% in the morning, 80-90% in the afternoon). Food habits among athletic adolescents are healthier and also are perceived as such in a higher proportion. Among athletic adolescents, consumption frequency is higher for dairy products and ready to eat (RTE) cereals, for fruit, fruit juices and salad (P<0.05 at least). Thus the athletic adolescent's food brings more micronutrients than the diet of their non-athletic counterparts. Within the subgroup (ancillary study), mean energy intake corresponds to requirements for age/gender group. CONCLUSIONS: Athletic adolescents display healthier food habits than non-athletic adolescents: this result supports the idea that healthy behavior tends to cluster and suggests that prevention programs among this age group should target simultaneously both sports activity and food habits.

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. II: Analysis of biological samples.

The potential and applicability of UHPSFC-MS/MS for anti-doping screening in urine samples were tested for the first time. For this purpose, a group of 110 doping agents with diverse physicochemical properties was analyzed using two separation techniques, namely UHPLC-MS/MS and UHPSFC-MS/MS in both ESI+ and ESI- modes. The two approaches were compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected, very diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a good complementarity of these analytical strategies. In both conditions, acceptable peak shapes and MS detection capabilities were obtained within 7min analysis time, enabling the application of these two methods for screening purposes. Method sensitivity was found comparable for 46% of tested compounds, while higher sensitivity was observed for 21% of tested compounds in UHPLC-MS/MS and for 32% in UHPSFC-MS/MS. The latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as signal suppression. In the case of UHPLC-MS/MS, more serious matrix effects were observed, leading typically to signal enhancement and the matrix effect was also concentration dependent, i.e., more significant matrix effects occurred at the lowest concentrations.

Food availability affects the maternal transfer of androgens and antibodies into eggs of a colonial seabird.

Mothers can improve the quality of their offspring by increasing the level of certain components in their eggs. To examine whether or not mothers increase deposition of such components in eggs as a function of food availability, we food-supplemented black-legged kittiwake females (Rissa tridactyla) before and during egg laying and compared deposition of androgens and antibodies into eggs of first and experimentally induced replacement clutches. Food-supplemented females transferred lower amounts of androgens and antibodies into eggs of induced replacement clutches than did non-food-supplemented mothers, whereas first clutches presented no differences between treatments. Our results suggest that when females are in lower condition, they transfer more androgens and antibodies into eggs to facilitate chick development despite potential long-term costs for juveniles. Females in prime condition may avoid these potential long-term costs because they can provide their chicks with more and higher quality resources.

Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

Clenbuterol is a β2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid-liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC-MS/MS with a linear gradient of acetonitrile in 9min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5pg/mL and a linearity range from 5pg/mL to 300pg/mL. Good trueness (85.8-105%), repeatability (5.7-10.6% RSD) and intermediate precision (5.9-14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10μg) of clenbuterol-enriched yogurts.

Seven years of gamma-ray spectrometry interlaboratory comparisons in Switzerland.

Since the 1990s, regular comparisons of gamma-ray spectrometry in Switzerland were organized to improve laboratory abilities to measure the radioactivity in the environment and food stuffs at typical routine levels. The activity concentration of the test samples and the evaluation of the associated uncertainties remained each year the main required test result. Over the years, the comparisons used certified reference solutions as well as environmental samples. The aim of this study is to research the effect of the comparisons on measurement quality. An analysis of the seven last interlaboratory comparisons revealed that the Swiss measurement capability is up to date. In addition, the results showed that the participants now have an improved evaluation of the uncertainties associated with their measurement.

Effects of the naturally occurring food mycotoxin ochratoxin A on brain cells in culture.

The potential of ochratoxin A (OTA) to damage brain cells was studied by using a three-dimensional cell culture system as model for the developing brain. Aggregating cell cultures of foetal rat telencephalon were tested either during an early developmental period, or during a phase of advanced maturation, over a wide range of OTA concentrations (0.4 nM to 50 microM). By monitoring changes in activities of cell type-specific enzymes (ChAt and GAD, for cholinergic and GABAergic neurones, respectively, GS for astrocytes and CNP for oligodendrocytes), the concentration-dependent toxicity and neurodevelopmental effects of OTA were determined. OTA proved to be highly toxic, since a 10-day treatment at 50 nM caused a general cytotoxicity in both mature and immature cultures. At 10 nM of OTA, cell type-specific effects were observed: in immature cultures, a loss in neuronal and oligodendroglial enzyme activities, and an increase in the activity of the astroglial marker glutamine synthetase were found, Furthermore, at 2 and 10 nM of OTA, a clustering of microglial cells was observed. In mature cultures, OTA was somewhat less potent, but caused a similar pattern of toxic effects. A 24 h-treatment with OTA resulted in a concentration-dependent decrease in protein synthesis, with IC50 values of 25 nM and 33 nM for immature and mature cultures respectively. Acute (24 h) treatment at high OTA concentrations (10 to 50 microM) caused a significant increase in reactive oxygen species formation, as measured by the intracellular oxidation of 2',7'-dichlorofluorescin. These results suggest that OTA has the potential to be a potent toxicant to brain cells, and that its effects at nanomolar concentrations are primarily due to the inhibition of protein synthesis, whereas ROS seem not to be involved in the toxicity mediated by a chronic exposure to OTA at such low concentrations.

Predicting positive food challenges in children sensitised to peanuts/tree nuts.

BACKGROUND: Children with atopic diseases in early life are frequently found with positive IgE tests to peanuts/tree nuts without a history of previous ingestion. We aimed to identify risk factors for reactions to nuts at first introduction. METHODS: A retrospective case-note and database analysis was performed. Recruitment criteria were: patients aged 3-16 yr who had a standardized food challenge to peanut and/or tree nuts due to sensitisation to the peanut/tree nut (positive spIgE or SPT) without previous consumption. A detailed assessment was performed of factors relating to food challenge outcome with univariate and multivariate logistic regression analysis. RESULTS: There were 98 food challenges (47 peanut, 51 tree nut) with 29 positive, 67 negative and 2 inconclusive outcomes. A positive maternal history of allergy and a specific IgE >5 kU/l were strongly associated with a significantly increased risk of a positive food challenge (OR 3.73; 95% CI 1.31-10.59; p = 0.013 and OR 3.35; 95% CI 1.23-9.11; p = 0.007, respectively). Adjusting for age, a three year-old with these criteria has a 67% probability of a positive challenge. There was no significant association between types of peanut/tree nut, other food allergies, atopic conditions or severity of previous food reactions and positive challenges. CONCLUSIONS: We have demonstrated an association between the presence of maternal atopic history and a specific IgE >5 kU/l, with a significant increase in the likelihood of a positive food challenge. Although requiring further prospective validation these easily identifiable components should be considered when deciding the need for a challenge.

Ecologic correlations of selected food groups with disease incidence and mortality in Switzerland.

Background: There is little information regarding the impact of diet on disease incidence and mortality in Switzerland. We assessed ecologic correlations between food availability and disease.Methods: In this ecologic study for the period 1970-2009, food availability was measured using the food balance sheets of the Food and Agriculture Organization of the United Nations. Standardized mortality rates (SMRs) were obtained from the Swiss Federal Office of Statistics. Cancer incidence data were obtained from the World Health Organization Health For All database and the Vaud Cancer Registry. Associations between food availability and mortality/incidence were assessed at lags 0, 5, 10, and 15 years by multivariate regression adjusted for total caloric intake.Results: Alcoholic beverages and fruit availability were positively associated, and fish availability was inversely associated, with SMRs for cardiovascular diseases. Animal products, meat, and animal fats were positively associated with the SMR for ischemic heart disease only. For cancer, the results of analysis using SMRs and incidence rates were contradictory. Alcoholic beverages and fruits were positively associated with SMRs for all cancer but inversely associated with all-cancer incidence rates. Similar findings were obtained for all other foods except vegetables, which were weakly inversely associated with SMRs and incidence rates. Use of a 15-year lag reversed the associations with animal and vegetal products, weakened the association with alcohol and fruits, and strengthened the association with fish.Conclusions: Ecologic associations between food availability and disease vary considerably on the basis of whether mortality or incidence rates are used in the analysis. Great care is thus necessary when interpreting our results.

Innovative method for carbon dioxide determination in human postmortem cardiac gas samples using headspace-gas chromatography-mass spectrometry and stable labeled isotope as internal standard.

A novel approach to measure carbon dioxide (CO2) in gaseous samples, based on a precise and accurate quantification by (13)CO2 internal standard generated in situ is presented. The main goal of this study was to provide an innovative headspace-gas chromatography-mass spectrometry (HS-GC-MS) method applicable in the routine determination of CO2. The main drawback of the GC methods discussed in the literature for CO2 measurement is the lack of a specific internal standard necessary to perform quantification. CO2 measurement is still quantified by external calibration without taking into account analytical problems which can often occur considering gaseous samples. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate in situ an internal labeled standard gas ((13)CO2) on the basis of the stoichiometric formation of CO2 by the reaction of hydrochloric acid (HCl) with sodium hydrogen carbonate (NaH(13)CO3). This method allows a precise measurement of CO2 concentration and was validated on various human postmortem gas samples in order to study its efficiency.

Indirect costs of parasitism are shaped by variation in the type of immune challenge and food availability

Parasites can inflict indirect fitness costs to their hosts by eliciting costly immune responses. These costs depend on the type and amount of immunostimulants presented to the host immune system but also on the amount of resources available to fuel host immune responses. Here, we investigated how the relative costs of two different types of immune challenge are modulated by variation in food availability. We injected nestling tawny owls (Strix aluco) with either 10 mu g of phytohaemagglutinin (PHA) or 20 mu g of lipopolysaccharide (LPS), and subsequently raised them under two different food regimes (food-restricted vs. ad libitum). After controlling for food consumption, we found that LPS-injected nestlings lost more body mass than PHA-injected ones only when food-restricted. We also found that body mass gain of owlets fed ad libitum decreased with the intensity of the skin swelling response against LPS, but not PHA. These experimental and correlative results suggest that nestling tawny owls suffered greater immune costs when treated with LPS than PHA, and that variation in the costs of two different types of immune challenge can be exacerbated under conditions of low food availability. Our study highlights the importance of taking into consideration the interplay between host immunity and nutrition in the study of indirect costs of parasitism.

Validation and long-term evaluation of a modified on-line chiral analytical method for therapeutic drug monitoring of (R,S)-methadone in clinical samples.

Matrix effects, which represent an important issue in liquid chromatography coupled to mass spectrometry or tandem mass spectrometry detection, should be closely assessed during method development. In the case of quantitative analysis, the use of stable isotope-labelled internal standard with physico-chemical properties and ionization behaviour similar to the analyte is recommended. In this paper, an example of the choice of a co-eluting deuterated internal standard to compensate for short-term and long-term matrix effect in the case of chiral (R,S)-methadone plasma quantification is reported. The method was fully validated over a concentration range of 5-800 ng/mL for each methadone enantiomer with satisfactory relative bias (-1.0 to 1.0%), repeatability (0.9-4.9%) and intermediate precision (1.4-12.0%). From the results obtained during validation, a control chart process during 52 series of routine analysis was established using both intermediate precision standard deviation and FDA acceptance criteria. The results of routine quality control samples were generally included in the +/-15% variability around the target value and mainly in the two standard deviation interval illustrating the long-term stability of the method. The intermediate precision variability estimated in method validation was found to be coherent with the routine use of the method. During this period, 257 trough concentration and 54 peak concentration plasma samples of patients undergoing (R,S)-methadone treatment were successfully analysed for routine therapeutic drug monitoring.

Nutrition transition in a middle-income country: 22-year trends in the Seychelles.

BACKGROUND/OBJECTIVES: There is little objective information regarding nutrition transition in African countries. We assessed trends in nutrition patterns in the Seychelles between 1989 and 2011. SUBJECTS/METHODS: Population-based samples were obtained in 1989, 1994 and 2011 and participants aged 25-44 are considered in this study (n=493, 599 and 471, respectively). Similar, although not identical, food frequency questionnaires (FFQs) were used in each survey and the variables were collapsed into homogenous categories for the purpose of this study. RESULTS: Between 1989 and 2011, consumption frequency of fish (5+/week) decreased from 93 to 74%, whereas the following increased: meat (5+/week) 25 to 51%, fruits (1+/week) 48 to 94%, salty snacks (1+/week) 22 to 64% and sweet snacks (1+/week) 38 to 67% (P<0.001 for all). Consumption frequency decreased for home-brewed alcoholic drinks (1+/week) 16 to 1%, but increased for wine (1+/week) 5 to 33% (both P<0.001). Between 2004 and 2011, consumption frequency decreased for rice (2/day) 62 to 57% and tea (1+/day) 72 to 68%, increased for poultry (1+/week) 86 to 96% (all P<0.01), and did not change for vegetables (70.3 to 69.8%, P=0.65). CONCLUSIONS: Seychelles is experiencing nutrition transition characterized by a decreased consumption frequency of traditional staple foods (fish, polished rice), beverages (tea) and of inexpensive home brews, and increased consumption frequency of meat, poultry and snacks. Food patterns also became more varied along with a broader availability of products in the 22-year interval. The health impact of these changes should be further studied.