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Langerak A.W.,Erasmus Medical Center | Groenen P.J.T.A.,Radboud University Nijmegen | Bruggemann M.,University of Kiel | Beldjord K.,Hopital St Louis | And 21 more authors.
Leukemia | Year: 2012

PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations. © 2012 Macmillan Publishers Limited.

Palmirotta R.,Laboratory of Molecular Diagnostics | De Marchis M.L.,Laboratory of Molecular Diagnostics | Ludovici G.,Laboratory of Molecular Diagnostics | Leone B.,Laboratory of Molecular Diagnostics | And 8 more authors.
International Journal of Biological Markers | Year: 2012

Multicenter studies and biobanking projects require blood transportation from the participating center to a central collection or diagnostic laboratory. The impact of time delays between venous blood collection and peripheral blood mononuclear cells (PBMC) isolation prior to RNA extraction may affect the quality and quantity of isolated nucleic acids for genomic applications. Thus, standard operating procedure (SOP) optimization for the treatment of biological samples before RNA extraction is crucial in a biological repository. In order to define SOPs for whole blood preservation prior to RNA extraction, we sought to determine whether different blood storage times (0, 3, 6, 10, 24, and 30 hours) prior to PBMCs isolation and storage at -80°C, could affect the quality and quantity of extracted RNA. After spectrophotometric quantification, the quality and integrity of RNA were assessed by agarose gel electrophoresis, RNA integrity number and real time-PCR (RT-PCR). Across the different time points we did not observe significant differences within the first 24 hours of blood storage at room temperature, while a significant loss in RNA yield and integrity was detected between 24 and 30 hours. We conclude that time delays before PBMCs isolation prior to RNA extraction may have a significant impact on downstream molecular biological applications. © 2012 Wichtig Editore.

Demidova I.,Laboratory of Molecular Diagnostics | Barinov A.,Laboratory of Molecular Diagnostics | Savelov N.,Moscow Oncological Hospital 62 | Gagarin I.,Laboratory of Molecular Diagnostics | And 8 more authors.
Archives of Pathology and Laboratory Medicine | Year: 2014

Context.-Echinoderm microtubule-associated proteinlike 4 gene (EML4) and anaplastic lymphoma kinase gene (ALK) fusion was shown to be the driver of tumorigenesis in approximately 3% to 5% of patients with non-small cell lung cancer (NSCLC) and is associated with response to inhibition with crizotinib. However, no complete agreement regarding the best diagnostic test for identification of ALK rearrangements has been achieved yet. Objective.-To investigate the concordance, sensitivity, and specificity of immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and reverse transcription- polymerase chain reaction (RT-PCR) for detection of ALK rearrangements. Design.-Thirty-six prospectively tested patients with NSCLC who had adenocarcinoma and 10 ALK-positive samples were included in the study. All samples were tested by IHC (ALK1 clone, 5A4 clone, D5F3 clone), FISH (LSI ALK Break Apart and ALK FISH Probe), and multiplexed RT-PCR. Results.- Immunohistochemistry staining was successful in all samples. Clone D5F3 showed the best sensitivity and specificity of 100%; clones ALK1 and 5A4 showed sensitivities of 91% with specificity of 100%. Both FISH probes showed concordance with sensitivity and specificity of 100%. Hybridization and RT-PCR were successful in 98% and 93.4% of samples, respectively, with sensitivity of 88% and specificity of 100%. Frequent artifacts leading to misinterpretation were observed with all 3 methodologies. Conclusions.-All 3 methodologies showed good sensitivity, specificity, and concordance, when artifacts were characterized and excluded. However, all ambiguous cases have to be confirmed as ALK rearranged by at least 2 of the 3 methods.

Van Lint P.,Laboratory of Molecular Diagnostics | Rossen J.W.,Laboratory of Medical Microbiology and Immunology | Vermeiren S.,Laboratory of Molecular Diagnostics | Ver Elst K.,Laboratory of Molecular Diagnostics | And 5 more authors.
Acta Clinica Belgica | Year: 2013

Diagnosis of intestinal parasites in stool samples is generally still carried out by microscopy; however, this technique is known to suffer from a low sensitivity and is unable to discriminate between certain protozoa. In order to overcome these limitations, a real-time multiplex PCR was evaluated as an alternative approach for diagnosing Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in stool samples. Therefore, a total of 631 faecal samples were analysed both by microscopy as well as by real-time PCR following automated DNA extraction. Results showed that real-time PCR exhibited sensitivity and specificity of both 100%, whereas traditional microscopy exhibited sensitivity and specificity of 37.5% and 99.8% respectively. As real-time PCR provides simple, sensitive and specific detection of these three important pathogenic protozoan parasites, this technique, rather than microscopy, has become our diagnostic method of choice for the detection of enteric protozoan parasites for the majority of patients.

De Sa L.R.V.,Brazilian National Institute of Technology | De Sa L.R.V.,Federal University of Rio de Janeiro | De Oliveira T.C.,Laboratory of Molecular Diagnostics | Dos Santos T.F.,Iguau University | And 4 more authors.
International Journal of Hydrogen Energy | Year: 2011

The conversion of organic compounds into H2 has received increasing attention. Enrichment of inocula by heat pretreatment eliminates non-spore forming hydrogen consuming microorganisms and promotes spore germination in genus Clostridium, which is known as one of the key hydrogen producers. Useful information about metabolic pathway is provided by some intermediate metabolites, such as: acetic, propionic, butyric and formic acids. The increase of acetic/butyric acids ratio indicates H2 production in heat pretreated inoculum when compared to untreated inoculum in the same cultivation conditions. The effect of heat pretreatment on inocula and consequently on the performance of bacterial communities responsible for H 2 production was monitored through the measurement of the level of hydrogenase gene expression, as well as through the content and distribution of volatile fatty acids. The acetic acid type fermentation was followed by the microorganisms presented in untreated and heat pretreated sludge. The medium containing untreated sludge presented a ratio of acetic/butyric acid of approximately 4, the same parameter was 7 when heat pretreated sludge was employed. The level of hydrogenase gene expression tripled when heat pretreated inoculum was used, indicating a higher production of H2. © 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights.

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