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.

T-Flux implant versus Healon GV in deep sclerectomy.

PURPOSE: To compare the efficacy and safety of T-Flux implant versus Healon GV in deep sclerectomy. METHODS: Randomized prospective trial of 23 eyes of 20 patients with medically uncontrolled open angle glaucoma over a period of 24 months, who underwent deep sclerectomy with either Healon GV or T-Flux implant. RESULTS: Mean postoperative intraocular pressure was 13.2 +/- 3.0 mm Hg with T-Flux implant (group 1) and 12.2 +/- 3.5 mm Hg with Healon GV (group 2), with a pressure reduction of 53.0% in group 1 (13.2 mm Hg vs. 28.1 mm Hg) and of 48.1% in group 2 (12.2 mm Hg vs. 23.5 mm Hg). Qualified and complete successes were 100% and 95.4% respectively. Pressures equal to or less than 15 mm Hg were 81.8% in group 1 and 90.9% in group 2 with or without treatment, and 63.6% in group 1 and 81.8% in group 2 without treatment. The number of glaucoma treatments dropped from 2.5 +/- 0.9 to 0.4 +/- 0.7 in group 1 and from 2.2 +/- 1.0 to 0.2 +/- 0.4 in group 2. The goniopuncture rate was 63.6% in group 1 and 36.4% in group 2, with a mean pressure drop of 6.1 +/- 3.9 mm Hg and 3.25 +/- 1.2 mm Hg respectively. Overall, slit-lamp diagnosed surgery-related complications included positive Seidel (13.6%), hyphaema (22.7%), choroidal detachment, and iris incarceration (4.5% each). At 2 years, ultrasound biomicroscopy showed mainly low reflective (40.1%) and flattened (36.4%) blebs. Principally latter ones were associated with the need for adjunctive treatment. A hypoechoic area in the suprachoroidal space was seen in at least 59.1% of eyes at 2 years and was not associated with lower intraocular pressure. CONCLUSION: Deep sclerectomy is an effective and safe surgery. However, longer follow up and larger study groups are required to assess the additional benefit of nonabsorbable implants.

Different degrees of ischaemic injury in the right and left ventricle in cases of severe, nonfatal, pulmonary embolism.

Pulmonary fat embolism (PFE) is a common complication of blunt force traumas with bone fractures. Severe forms cause impedance to right ventricular (RV) ejection, with eventual right heart ischaemia and failure. In a prospective study, we have investigated 220 consecutive autopsy cases (73 females, 147 males, mean age 52.1 years, min 14 years, max 91 years). PFE was detected in 52 cases that were divided into three groups according to the degree of PFE (1-3). A fourth group of cases of violent death without PFE was used for comparison. In each case, histology (H&E, Masson) and immunohistochemistry (fibronectin and C5b-9) were performed on six cardiac samples (anterior, lateral and posterior wall of both ventricles). The degree of cardiac damage was registered in each sample and the mean degree of damage was calculated in each case at the RV and left ventricle (LV). Moreover, a parameter ∆ that is the difference between the mean damage at the RV and the LV was calculated in each case. The results were compared within each group and between the groups. In the present study, we could not detect prevalent RV damage in cases of high degree PFE as we did in our previous investigation. In the group PFE3 the difference of the degree of damage between the RV and LV was higher than the one observed in the groups PFE0-2 with the antibody anti-fibronectin. Prevalent right ventricular stress in cases of severe PFE may explain this observation.

Endovascular treatment of pulmonary arteriovenous malformations in hereditary haemorrhagic telangiectasia.

PRINCIPLES: To assess the efficiency and complication rates of vaso-occlusion of pulmonary arteriovenous malformations (PAVMs) in Rendu-Osler-Weber disease (hereditary haemorrhagic telangectasia; HHT). METHODS: Seventy-two patients were investigated in our institution for HHT between March 2000 and November 2011. Sixteen presented PAVMs (22.2%), and 11 (68.8%) were treated with vaso-occlusion for a total of 18 procedures. Procedures included coils, plugs and combined approaches. Immediate success and recurrence rate, complication were recorded, as well as persistent and new PAVMs during clinical and computed tomography (CT) follow-up. RESULTS: Eighteen procedures were performed and a total of 37 PAVMs were treated, 19 with coils, 16 with plugs and 2 with combined treatment. Mean CT follow-up time was 41 months (1‒164). No major complication was observed. One distal translocation was treated during the same intervention. Two PAVMs persisted after treatment (5.7%), both treated by means of plug embolisation. One new PAVM was observed during follow-up CT. PAVMs with an afferent artery of less than 3mm or asymptomatic PAVMs were not treated. CONCLUSION: Recent studies have demonstrated that vaso-occlusion has become the gold standard treatment for PAVM. This study is in accordance with previous results and shows a minimal complication rate and little recurrence, whether by coils, plugs, or combined treatments.

On the rationale of population screening for chronic kidney disease : a public health perspective

Chronic kidney disease (CKD) and its complications represent an enormous and increasing public health burden worldwide [1]. More than one in ten adults suffers from CKD in the general population [2], with a majority of people being in its early stages (i.e. 1 to 3) [2]. In the general population, the prevalence of CKD sharply increases with age [3]. CKD can be considered as a condition associated with premature ageing with accelerated vascular disease [4]. The large number of people with CKD, or at high risk of CKD (i.e. patients with hypertension, diabetes and/or CVD), implies that primary care providers and specialists other than nephrologists frequently encounter patients with CKD [5], a situation in which most CKD cases are diagnosed via opportunistic kidney function screening or automated eGFR reporting. The aim of this review is to discuss the rationale and currently available evidence for, or against, population-based screening for CKD. The focus will be on the situation of screening asymptomatic individuals at early stages of CKD regardless of the presence or absence of CKD risk factors.

Fast spin echo imaging of carotid artery dynamics.

PURPOSE: We propose the use of a retrospectively gated cine fast spin echo (FSE) sequence for characterization of carotid artery dynamics. The aim of this study was to compare cine FSE measures of carotid dynamics with measures obtained on prospectively gated FSE images. METHODS: The common carotid arteries in 10 volunteers were imaged using two temporally resolved sequences: (i) cine FSE and (ii) prospectively gated FSE. Three raters manually traced a common carotid artery area for all cardiac phases on both sequences. Measured areas and systolic-diastolic area changes were calculated and compared. Inter- and intra-rater reliability were assessed for both sequences. RESULTS: No significant difference between cine FSE and prospectively gated FSE areas were observed (P201;=201;0.36). Both sequences produced repeatable cross-sectional area measurements: inter-rater intraclass correlation coefficient (ICC)201;=201;0.88 on cine FSE images and 0.87 on prospectively gated FSE images. Minimum detectable difference (MDD) in systolic-diastolic area was 4.9 mm(2) with cine FSE and 6.4 mm(2) with prospectively gated FSE. CONCLUSION: This cine FSE method produced repeatable dynamic carotid artery measurements with less artifact and greater temporal efficiency compared with prospectively gated FSE. Magn Reson Med 74:1103-1109, 2015. © 2014 Wiley Periodicals, Inc.

The VZV/IE63-specific T cell response prevents herpes zoster in fingolimod-treated patients.

OBJECTIVE: To assess longitudinally the antiviral immune response of T cells from patients with multiple sclerosis (MS) treated with fingolimod (FTY) vs other disease-modifying treatments (DMTs). METHODS: We assessed cellular immune responses specific to influenza virus (FLU), JC virus (JCV), and varicella-zoster virus (VZV) using quantification of interferon-γ secretion by enzyme-linked immunospot in patients with MS on FTY (n = 31), including 2 with herpes zoster (HZ), natalizumab (n = 11), and other DMTs (n = 11). We used viral lysates for FLU and VZV and a pool of peptides for FLU, JCV (VP-1), and VZV (IE63). RESULTS: Besides an expected drop of T cells, we found that, proportionally to the number of CD3(+) T cells, only FTY-treated patients with MS exhibited an increased VZV/IE63-specific T cell response peaking 6 months into treatment, a response that returned to baseline after 12 and 24 months. Two FTY-treated patients developed an HZ 6 months into treatment, coinciding with an absent VZV/IE63-specific T cell response. However, cellular immune responses specific to VZV lysate, JCV, and FLU (lysate and pool of peptide epitopes) were similar between all 3 categories (FTY, natalizumab, and other DMTs) of study patients. CONCLUSIONS: FTY-treated patients with MS exhibit an increased VZV/IE63-specific cellular immune response after 6 months of treatment. FTY-treated patients who develop an HZ are not able to mount such a response, suggesting that a T cell response directed against this viral protein may be key in preventing the occurrence of HZ.

Sleep Characteristics in Early Stages of Chronic Kidney Disease in the HypnoLaus Cohort.

STUDY OBJECTIVES: To evaluate the association between early stages of chronic kidney disease (CKD) and sleep disordered breathing (SDB), restless legs syndrome (RLS), and subjective and objective sleep quality (SQ). METHODS: Cross-sectional analysis of a general population-based cohort (HypnoLaus). 1,760 adults (862 men, 898 women; age 59.3 (± 11.4) y) underwent complete polysomnography at home. RESULTS: 8.2% of participants had mild CKD (stage 1-2, estimated glomerular filtration rate [eGFR] ≥ 60 mL/min/1.73 m(2) with albuminuria) and 7.8% moderate CKD (stage 3, eGFR 30-60 mL/min/1.73 m(2)). 37.3% of our sample had moderate-to-severe SDB (apnea-hypopnea index [AHI] ≥ 15/h) and 15.3% had severe SDB (AHI ≥ 30/h). SDB prevalence was positively associated with CKD stages and negatively with eGFR. In multivariate analysis, age, male sex, and body mass index were independently associated with SDB (all P < 0.001), but kidney function was not. The prevalence of RLS was 17.5%, without difference between CKD stages. Periodic leg movements index (PLMI) was independently associated with CKD stages. Subjective and objective SQ decreased and the use of sleep medication was more frequent with declining kidney function. Older age, female sex, and the severity of SDB were the strongest predictors of poor SQ in multivariate regression analysis but CKD stage was also independently associated with reduced objective SQ. CONCLUSIONS: Patients with early stages of CKD have impaired SQ, use more hypnotic drugs, and have an increased prevalence of SDB and PLM. After controlling for confounders, objective SQ and PLMI were still independently associated with declining kidney function.

Determinação em fluxo do carbonatoresidual do solo empregando pervaporação

The objective of this study was to develop a method for the direct determination of residual CO3(2-) in acid soil using a flow system coupled with pervaporation. The gas released from the acidified sample was separated though a hydrophobic membrane and detected by conductivimetry. The detection limit was 0.054 mmol c kg-1 for CO2/CO3(2-) with relative error of 3.1%. The analytical frequency was 20 samples per hour. The method is recommended for studying the reactions and kinetics of lime applied in acid soils.

A synthetic approach to palmerolides via Negishi cross coupling. The challenge of the C15-C16 bond formation

The esterification of fragment C1-C8 (2) with fragment C16-C23 (3) to give iodo derivative 4, followed by a Pd-catalysed coupling with a C9-C15 fragment (7 or 8), may provide a common precursor of most palmerolides. Ligands and reaction conditions were exhaustively examined to perform the C15-C16 bond formation via Negishi reaction. With simple models, pre-activated Pd-Xantphos and Pd-DPEphos complexes were the most efficient catalysts at RT. Zincation of the C9-C15 fragment (8) and cross coupling with 4 required 3 equiv of t-BuLi, 10 mol % of Pd-Xantphos and 60 °C.

Building complexity in O2-binding copper complexes : site-selective metalation and intermolecular O2-binding at dicopper and heterometallic complexes derived from an unsymmetric ligand

A novel unsymmetric dinucleating ligand (LN3N4) combining a tridentate and a tetradentate binding sites linked through a m-xylyl spacer was synthesized as ligand scaffold for preparing homo- and dimetallic complexes, where the two metal ions are bound in two different coordination environments. Site-selective binding of different metal ions is demonstrated. LN3N4 is able to discriminate between CuI and a complementary metal (M′ = CuI, ZnII, FeII, CuII, or GaIII) so that pure heterodimetallic complexes with a general formula [CuIM′(LN3N4)]n+ are synthesized. Reaction of the dicopper(I) complex [CuI 2(LN3N4)]2+ with O2 leads to the formation of two different copper-dioxygen (Cu2O2) intermolecular species (O and TP) between two copper atoms located in the same site from different complex molecules. Taking advantage of this feature, reaction of the heterodimetallic complexes [CuM′(LN3N4)]n+ with O2 at low temperature is used as a tool to determine the final position of the CuI center in the system because only one of the two Cu2O2 species is formed

Triggering the generation of an iron(IV)-oxo compound and its reactivity toward sulfides by RuII photocatalysis

The preparation of [FeIV(O)(MePy2tacn)]2+ (2, MePy2tacn = N-methyl-N,N-bis(2-picolyl)-1,4,7-triazacyclononane) by reaction of [FeII(MePy2tacn)(solvent)]2+ (1) and PhIO in CH3CN and its full characterization are described. This compound can also be prepared photochemically from its iron(II) precursor by irradiation at 447 nm in the presence of catalytic amounts of [Ru II(bpy)3]2+ as photosensitizer and a sacrificial electron acceptor (Na2S2O8). Remarkably, the rate of the reaction of the photochemically prepared compound 2 toward sulfides increases 150-fold under irradiation, and 2 is partially regenerated after the sulfide has been consumed; hence, the process can be repeated several times. The origin of this rate enhancement has been established by studying the reaction of chemically generated compound 2 with sulfides under different conditions, which demonstrated that both light and [Ru II(bpy)3]2+ are necessary for the observed increase in the reaction rate. A combination of nanosecond time-resolved absorption spectroscopy with laser pulse excitation and other mechanistic studies has led to the conclusion that an electron transfer mechanism is the most plausible explanation for the observed rate enhancement. According to this mechanism, the in-situ-generated [RuIII(bpy)3] 3+ oxidizes the sulfide to form the corresponding radical cation, which is eventually oxidized by 2 to the corresponding sulfoxide

Otimização das condições de produção de ésteres etílicos a partir de óleo de peixe com elevado teor de ácidos graxos ômega-3

The production of ethyl esters by alcoholysis is an alternative for splitting triacylglycerols due to the possibility of using low temperatures, which results in oxidative protection of the polyunsaturated fatty acids. Ethyl esters produced under mild conditions of temperature could be used as substrate for obtaining structured lipids. The reaction parameters of production of ethyl esters from fish oil with high content of omega-3 fatty acids by alcoholysis were optimized using response surface methodology. An experimental design (2³) (with levels +1 and -1, six axial points with levels -alpha and +alpha and three central points) was applied. The variables investigated were concentration of catalyst, amount of ethyl alcohol and temperature. Ethyl ester conversion was monitored by high performance size exclusion chromatography (HPSEC) and the best result obtained was 95% conversion rate. The optimal conditions were 40 °C, 1% of NaOH and 36% of ethanol.

Quantificação de sulfadimetoxina em leite por cromatografia líquida de alta eficiência

A method for HPLC determination of sulfadimethoxyne in milk is presented. The analyte isolation and concentration were performed by solid-phase extraction through a C-8 cartridge, pre-conditioned with hexane, methanol and water and eluted with MeOH. The recovery determination was done with a spiked solution of 20, 50 or 100 µg L-1. In this concentration range, the recovery was 83.2% with a RDS of 15.4%. For quantification, a Zorbax Eclipse XDB-C8 (4.6 mm x 150 mm, 5 µm), a mobile phase of MeCN: 0.01 mol L-1 KH2PO4 aq. (1:4), and a variable wavelength detector (275 nm) were used.

Development and validation of a dissolution test for telithromycin in coated tablets

A dissolution test for telithromycin tablets was validated and developed. In order to choose the most discriminatory one, the conditions to carry out are 900 mL of sodium phosphate buffer at pH 7.5, paddles at 50 rpm stirring speed, time test set to 60 min and using USP apparatus 2 with paddles. The UV spectrophotometric method for determination of telithromycin released was developed and validated. The method presents linearity (r = 1) in the concentration range of 20-60 µg/mL. Precision and recoveries were good, 100.62 and 97.06%, respectively. The method was successfully used for the dissolution test of telithromycin tablets.