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1.
Acta Cytol ; 65(1): 88-98, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33011718

RESUMO

OBJECTIVE: Although transcriptomic assessments of small samples using high-throughput techniques are usually performed on fresh or frozen tissues, there is a growing demand for those performed on stained cellular specimens already used for diagnostic purposes. STUDY DESIGN: The possibility of detecting mRNAs and microRNAs (miRNAs) from routinely processed cytological samples using nCounter® technology was explored. Fresh samples from pleural and peritoneal effusions were analyzed using 2 parallel methods: samples were smeared and routinely stained using the May-Grünwald-Giemsa or Diff-Quik® method and mounted using conventional methods, and they were also studied following a snap freezing method, in which samples were maintained at -80°C until use. mRNAs and miRNAs were assessed and compared after total RNA extraction from both routinely processed samples and their matched frozen controls. RESULTS: A good concordance was found between the gene expression measured in routinely processed samples and their matched frozen controls for the majority of mRNAs and miRNAs tested. However, the standard deviation of low-expressed miRNA was high. CONCLUSIONS: Although nCounter® technology is a robust method to measure and characterize both mRNAs and miRNAs from routinely processed cytological samples, caution is recommended for the interpretation of low-expressed miRNA.


Assuntos
Técnicas Citológicas/métodos , Perfilação da Expressão Gênica/métodos , Expressão Gênica/genética , MicroRNAs/genética , RNA Mensageiro/genética , Corantes Azur/química , Amarelo de Eosina-(YS)/química , Humanos , Azul de Metileno/química , Inclusão em Parafina/métodos , Estudo de Prova de Conceito , Xantenos/química
2.
Methods Mol Biol ; 1541: 189-208, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27910025

RESUMO

Cytogenetics is the gold-standard in biological dosimetry for assessing a received dose of ionizing radiation. More modern techniques have recently emerged, but none are as specific as cytogenetic approaches, particularly the dicentric assay. Here, we will focus on the principal cytogenetic techniques used for biological dosimetry: the dicentric assay in metaphase cells, the micronuclei assay in binucleated cells, and the premature condensed chromosome (PCC) assay in interphase cells. New fluorescence in situ hybridization (FISH) techniques (such as telomere-centromere hybridization) have facilitated the analysis of the dicentric assay and have permitted to assess the dose a long time after irradiation by translocation analysis (such as by Tri-color FISH or Multiplex-FISH). Telomere centromere staining of PCCs will make it possible to perform dose assessment within 24 h of exposure in the near future.


Assuntos
Aberrações Cromossômicas/efeitos da radiação , Análise Citogenética/métodos , Radiação Ionizante , Radiometria/métodos , Animais , Relação Dose-Resposta à Radiação , Humanos , Micronúcleos com Defeito Cromossômico , Microscopia , Translocação Genética
3.
Int J Radiat Biol ; 93(1): 75-80, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27559844

RESUMO

PURPOSE: The RENEB accident exercise was carried out in order to train the RENEB participants in coordinating and managing potentially large data sets that would be generated in case of a major radiological event. MATERIALS AND METHODS: Each participant was offered the possibility to activate the network by sending an alerting email about a simulated radiation emergency. The same participant had to collect, compile and report capacity, triage categorization and exposure scenario results obtained from all other participants. The exercise was performed over 27 weeks and involved the network consisting of 28 institutes: 21 RENEB members, four candidates and three non-RENEB partners. RESULTS: The duration of a single exercise never exceeded 10 days, while the response from the assisting laboratories never came later than within half a day. During each week of the exercise, around 4500 samples were reported by all service laboratories (SL) to be examined and 54 scenarios were coherently estimated by all laboratories (the standard deviation from the mean of all SL answers for a given scenario category and a set of data was not larger than 3 patient codes). CONCLUSIONS: Each participant received training in both the role of a reference laboratory (activating the network) and of a service laboratory (responding to an activation request). The procedures in the case of radiological event were successfully established and tested.


Assuntos
Planejamento em Desastres/organização & administração , Monitoramento de Radiação/métodos , Liberação Nociva de Radioativos , Radiobiologia/educação , Gestão da Segurança/organização & administração , Triagem/organização & administração , Europa (Continente)
4.
Int J Radiat Biol ; 93(1): 2-14, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27707245

RESUMO

PURPOSE: A European network was initiated in 2012 by 23 partners from 16 European countries with the aim to significantly increase individualized dose reconstruction in case of large-scale radiological emergency scenarios. RESULTS: The network was built on three complementary pillars: (1) an operational basis with seven biological and physical dosimetric assays in ready-to-use mode, (2) a basis for education, training and quality assurance, and (3) a basis for further network development regarding new techniques and members. Techniques for individual dose estimation based on biological samples and/or inert personalized devices as mobile phones or smart phones were optimized to support rapid categorization of many potential victims according to the received dose to the blood or personal devices. Communication and cross-border collaboration were also standardized. To assure long-term sustainability of the network, cooperation with national and international emergency preparedness organizations was initiated and links to radiation protection and research platforms have been developed. A legal framework, based on a Memorandum of Understanding, was established and signed by 27 organizations by the end of 2015. CONCLUSIONS: RENEB is a European Network of biological and physical-retrospective dosimetry, with the capacity and capability to perform large-scale rapid individualized dose estimation. Specialized to handle large numbers of samples, RENEB is able to contribute to radiological emergency preparedness and wider large-scale research projects.


Assuntos
Bioensaio/métodos , Planejamento em Desastres/organização & administração , Lesões por Radiação/prevenção & controle , Monitoramento de Radiação/métodos , Proteção Radiológica/métodos , Gestão da Segurança/organização & administração , Emergências , Europa (Continente) , Humanos , Objetivos Organizacionais , Exposição à Radiação/análise , Exposição à Radiação/prevenção & controle , Liberação Nociva de Radioativos/prevenção & controle
5.
Sci Rep ; 6: 32510, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27587191

RESUMO

Telomeres are specific structures that protect chromosome ends and act as a biological clock, preventing normal cells from replicating indefinitely. Mammalian telomeres are replicated throughout S-phase in a predetermined order. However, the mechanism of this regulation is still unknown. We wished to investigate this phenomenon under physiological conditions in a changing environment, such as the immortalization process to better understand the mechanism for its control. We thus examined the timing of human telomere replication in normal and SV40 immortalized cells, which are cytogenetically very similar to cancer cells. We found that the timing of telomere replication was globally conserved under different conditions during the immortalization process. The timing of telomere replication was conserved despite changes in telomere length due to endogenous telomerase reactivation, in duplicated homologous chromosomes, and in rearranged chromosomes. Importantly, translocated telomeres, possessing their initial subtelomere, retained the replication timing of their homolog, independently of the proportion of the translocated arm, even when the remaining flanking DNA is restricted to its subtelomere, the closest chromosome-specific sequences (inferior to 500 kb). Our observations support the notion that subtelomere regions strongly influence the replication timing of the associated telomere.


Assuntos
Período de Replicação do DNA , Telômero/metabolismo , Linhagem Celular Transformada , Cromossomos Humanos/genética , Fibroblastos/metabolismo , Humanos , Modelos Biológicos , Fase S , Transfecção
6.
Cancer Cytopathol ; 124(4): 241-53, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26505273

RESUMO

BACKGROUND: Data generated by next-generation sequencing technologies have a pivotal role in precision medicine. These high-throughput techniques are preferentially performed on fresh tissue, but there is an increasing need for protocols adapted to materials derived from formalin-fixed, paraffin-embedded tissue and cytology specimens. METHODS: The aim of this work was to show that cytological material collected from archival smears processed for routine diagnoses could be used for massively parallel sequencing and array-based genomic analysis for further studies. RESULTS: As a proof of concept, data obtained from May-Grünwald Giemsa- and Diff-Quik-stained archival smears were shown to be in keeping with those obtained from matched frozen controls. CONCLUSIONS: The quality of DNA extracted from routinely processed smears is compatible with the multitargeted sequencing of a large series of genes of interest with methods such as array-based genomic analysis and whole-exome sequencing.


Assuntos
DNA de Neoplasias/genética , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Polimorfismo de Nucleotídeo Único/genética , Medicina de Precisão , Análise de Sequência de DNA/métodos , Estudos de Coortes , Citodiagnóstico/métodos , Testes Diagnósticos de Rotina , Humanos , Neoplasias/patologia , Inclusão em Parafina , Reação em Cadeia da Polimerase/métodos , Estudos Retrospectivos , Sensibilidade e Especificidade
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