RESUMO
We aimed to directly align a chromosomal CGH (cCGH) pattern with the gene mapping data by taking advantage of the clustering of the GGCC motif at certain positions in the human genome. The alignment of chromosomal with sequence data was achieved by superimposition of (i) the fluorescence intensity of the sequence specific fluorochrome, Chromomycin A3 (CMA3), (ii) the cCGH fluorescence intensity profile of individual chromosomes and (iii) the GGCC density profile extracted from the Ensembl genome sequence database. The superimposition of these three pieces of information allowed us to precisely localize regions of amplification in the neuroblastoma cell line STA-NB-15. Two prominent cCGH peaks were noted, one at 2p24.3, the position 15.4 mega base (Mb), and the other at 2p23.2, 29.51 Mb. FISH and high resolution array CGH (aCGH) experiments disclosed an amplification of MYCN (16 Mb) and ALK (29.2-29.9 Mb), thus confirming the cCGH data. The combined visualization of sequence information and cCGH data drastically improves the resolution of the method to less than 2 Mb.
Assuntos
Mapeamento Cromossômico/métodos , Hibridização de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Quinase do Linfoma Anaplásico , Sequência de Bases , Linhagem Celular Tumoral , Cromomicina A3 , Citogenética/métodos , DNA/genética , DNA de Neoplasias/genética , Corantes Fluorescentes , Genoma Humano , Humanos , Hibridização in Situ Fluorescente/métodos , Proteína Proto-Oncogênica N-Myc , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Proteínas Tirosina Quinases/genética , Receptores Proteína Tirosina QuinasesRESUMO
The quantification of DNA damage, both in vivo and in vitro, can be very time consuming, since large amounts of samples need to be scored. Additional uncertainties may arise due to the lack of documentation or by scoring biases. Image analysis automation is a possible strategy to cope with these difficulties and to generate a new quality of reproducibility. In this communication we collected some recent results obtained with the automated scanning platform Metafer, covering applications that are being used in radiation research, biological dosimetry, DNA repair research and environmental mutagenesis studies. We can show that the automated scoring for dicentric chromosomes, for micronuclei, and for Comet assay cells produce reliable and reproducible results, which prove the usability of automated scanning in the above mentioned research fields.
Assuntos
Análise Citogenética/instrumentação , Processamento de Imagem Assistida por Computador/instrumentação , Microscopia/métodos , Animais , Automação , Contagem de Células/instrumentação , Núcleo Celular/ultraestrutura , Aberrações Cromossômicas , Ensaio Cometa/instrumentação , Dano ao DNA , Desenho de Equipamento , Raios gama , Humanos , Linfócitos/efeitos da radiação , Linfócitos/ultraestrutura , Microcomputadores , Testes para Micronúcleos/instrumentação , Microscopia/instrumentação , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Testes de Mutagenicidade/instrumentação , Radiometria/instrumentação , Radiometria/métodos , Reprodutibilidade dos TestesRESUMO
We combined laser-assisted microdissection from H&E-stained paraffin sections, degenerated oligonucleotide-primed polymerase chain reaction (DOP-PCR), and comparative genomic hybridization (CGH) to analyse chromosomal imbalances in small tumour areas consisting of 50-100 cells. This approach was used to investigate intratumour genetic heterogeneity in a case of metastatic prostatic adenocarcinoma and chromosomal changes in areas of prostatic intraepithelial neoplasia (PIN) adjacent to the invasive tumour. In four microdissected invasive tumour areas with different histological patterns (acinar, cribriform, papillary and solid) marked intratumour heterogeneity was found by CGH. Recurrent chromosomal imbalances detected in at least two microdissected tumour areas were gains on 1p32-->p36, 2p22, 3q21, 7, 8q21-->q24, 11q12-->q13, 16p12-->p13, 17, 19 and loss on 16q23. Additional chromosomal changes were found in only one of the microdissected areas (gains on 16q21-->q23, 20q22 and losses on 8p21-->p23, 12p11-->q12, 12q21-->q26, 13q21-->q34, 16q12, and 18q22). In PIN, gains on chromosomes 8q21-->q24 and 17 were found in both samples investigated (low and high grade PIN), while gains on chromosomes 7, 11q, 12q, 16p, and 20q and losses on 2p, 8p21-->p23, 12q were found only in one PIN area. Controls to ensure reliable CGH results consisted in CGH analyses of (i) approximately 80 microdissected normal epithelial cells, which showed no aberrations after DOP-PCR and (ii) larger cell numbers (approximately 10(5) or 10(7) cells) of the primary tumour investigated without DOP-PCR and partially displaying the chromosomal imbalances (gain on 16p12-->p13, losses on 2p25, 8p21-->p23, 12p11-->p12, 12q21-->q26, 18q22) found in the small microdissected areas. Microsatellite and FISH analyses further confirmed our CGH results from microdissected cells. The combined approach of laser-assisted microdissection, DOP-PCR and CGH is suitable to identify early genetic changes in PIN and chromosomal imbalances associated with the particular histological patterns of invasive prostatic adenocarcinoma.
Assuntos
Adenocarcinoma/genética , DNA de Neoplasias/análise , Neoplasia Prostática Intraepitelial/genética , Neoplasias da Próstata/genética , Adenocarcinoma/secundário , Idoso , Mapeamento Cromossômico , Primers do DNA/química , Técnicas de Preparação Histocitológica , Humanos , Hibridização in Situ Fluorescente/métodos , Cariotipagem , Metástase Linfática/genética , Metástase Linfática/patologia , Masculino , Repetições de Microssatélites/genética , Reação em Cadeia da Polimerase , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/patologiaRESUMO
BACKGROUND: The reliable detection and quantification of gene amplifications is crucial to clinical practice. Although there are different detection techniques, the fluorescence in situ hybridization (FISH) method has become highly accepted over past years because it is a reliable, robust, and quick method. Unfortunately, automatic quantification of gene amplification based on fluorescence intensities has not been possible thus far. Because current spot counting methods are reliable only when analyzing low amplification rates, we attempted to establish another method, i.e., to quantify the intensity of different FISH signals using an automatic fluorescence microscopical device on interphase nuclei: interphase quantitative FISH (IQ-FISH). METHODS: We quantified the fluorescence intensities of the differently labeled FISH probes (MYCN and D2Z) hybridized to three different neuroblastoma cell lines, six peripheral blood (PB) samples, 10 spiked PB samples, and nine neuroblastoma samples using the Metafer4 system (MetaSystems, Altlussheim, Germany). To obtain the MYCN copy number per cell, the ratio between the fluorescence intensities of the MYCN gene and reference sequence (D2Z) was calculated. For automatic analysis of the HER-2/neu status in tumor cells, labeled FISH probes specific for HER-2/neu and a chromosome 17-specific probe were hybridized to peripheral blood and tumor specimens and analyzed using the automatic device. RESULTS: When measuring the fluorescence intensity per cell for both probe pairs (MYCN/D2Z and HER-2/17p), amplified and non-amplified cells, showed distinct peaks with only little overlap. Whereas normal cells showed a fluorescence ratio peak for MYCN/D2Z between 200 and 800, cells with MYCN amplification clearly exceeded this ratio value (1000 to 25,000). When mixing a varying number of MYCN amplified cells (range 9-91%) to normal PB, the spiked tumor cells could be identified. Even one neuroblastoma tumor cell in 1000 mononucleated cells could reliably be detected using our device. In neuroblastoma patient samples, non-amplified cells were distinguished from amplified cells. Automatically and manually counted signals gave matching results in amplified and non-amplified samples. HER-2/neu-amplified cells were automatically detected in the breast cancer samples analyzed. CONCLUSION: The automatic measurement of fluorescence signal intensities not only allows a reliable discrimination between non-amplified and amplified cells but also exact quantification of amplified sequences. This is the prerequisite for the following applications: detection of amplified cells in the bone marrow and second-look specimens; comparison between primary and relapse or pre- and post-chemotherapeutic specimens; detection of tumors with focal gene amplification; and quantification of elimination of amplified gene sequences.
Assuntos
Amplificação de Genes , Hibridização in Situ Fluorescente/métodos , Automação/métodos , Linhagem Celular Tumoral , Humanos , NeuroblastomaRESUMO
The efficiency of the automated metaphase finding system METAFER2 is assessed in a routine mutagenicity assay using an aneuploid rat liver cell line treated with various promutagens. Data sets generated by automated and manual selection of metaphases are compared. It is demonstrated that METAFER2 routinely allows an efficient automatic identification of metaphases not only in lymphocyte preparations, but also in preparations from mammalian cell lines with varying chromosome numbers. Although larger slide areas are required for automated compared to manual metaphase scanning, the automatic system is faster by a factor of about 5. The interactive visual elimination of metaphases of insufficient quality is an easy and fast procedure. METAFER2 allows an unbiased selection of metaphases irrespective of their appearance as homogeneously stained first or harlequin-stained second division cells. Random selection of metaphases is neither influenced by various structural chromosome changes nor by increased frequencies of sister-chromatid exchanges.
Assuntos
Processamento de Imagem Assistida por Computador , Metáfase , Testes de Mutagenicidade/métodos , Aneuploidia , Animais , Automação , Linhagem Celular , Aberrações Cromossômicas , Estudos de Avaliação como Assunto , Mutagênicos/toxicidade , Ratos , Troca de Cromátide Irmã , Fatores de TempoRESUMO
Based on the technical configuration of the Metafer2 metaphase finder (Metasystems, Altlussheim, Germany), an automated system was developed that is able to discriminate first/M1 or second cycle/M2 metaphases. For an evaluation of the system in fluorescence plus Giemsa stained preparations of human lymphocytes, a learning sample and an independent test sample was used. Between 8 and 14 separated chromosomes per metaphase were sufficient to achieve correct classification rates between 95 and 100%. For practical application the system can be most efficiently used for biodosimetry of human radiation exposure which requires scoring of thousands of metaphases exclusively in M1. An application for selection of M2 for SCE scoring in mutagenicity testing was possible, but does not provide a comparable advantage.
Assuntos
Aberrações Cromossômicas , Técnicas Genéticas , Processamento de Imagem Assistida por Computador , Metáfase , Corantes Azur , Classificação , Fluorescência , HumanosRESUMO
The Metafer2 fluorescence scanning system was used for routine analysis of radiation-induced exchange aberrations measured by fluorescence in situ hybridisation (FISH) chromosome painting in human peripheral lymphocytes. The system enables a rapid and unbiased fully-automated finding and image acquisition of fluorescently stained metaphase spreads. The chromosome aberration analysis is performed interactively from stored digitised processed gallery images, presented on a screen. Appropriate software image filters are available to further improve these pictures by background correction, noise reduction and fluorescence signal enhancement. Data sets generated by computer-assisted and manual scoring of radiation-induced reciprocal translocations (2B) and total 2B (2B+related 'one-way' types) or complete dicentrics (2A) and total 2A (2A+related 'one-ways') involving painted target chromosomes 2, 3 or 4 were compared and no significant differences were found.A linear-quadratic dose-response curve for total translocations (2B+'one-ways'+complex-derived types) based on computer-assisted analysis of 27,741 metaphases with chromosome 4 painting was compared to a curve obtained earlier for manually scored translocations in a set of target chromosomes 1, 4 and 12. After extrapolation to the whole genome, no significant difference between both curves was found. From our results it can be derived that computer-assisted aberration analysis using the Metafer2 system is a reliable alternative to manual analysis. Since time saving for computer-assisted translocation analysis is about 50% compared to manual scoring, this system is highly promising for a practical application in retrospective biodosimetry of human radiation exposure.
Assuntos
Coloração Cromossômica/métodos , Processamento de Imagem Assistida por Computador , Linfócitos/efeitos da radiação , Metáfase/efeitos da radiação , Testes de Mutagenicidade/métodos , Translocação Genética/efeitos da radiação , Adulto , Automação , Células Cultivadas/citologia , Relação Dose-Resposta à Radiação , Feminino , Humanos , Modelos Lineares , Linfócitos/citologia , MasculinoRESUMO
Accurate quantification of disseminated tumor cells in hematological samples is of fundamental importance in clinical oncology. However, even highly standardized protocols allow only a rough estimation of the total analyzed cell number, as sample processing may have adverse effects on the number of cells available for analysis. The fluorescence-based microscopic scanning system (MetaCyte) detects, counts, captures, and relocates immunolabeled tumor cells in hematopoietic samples. We report on a cell-counting approach that has been implemented into the scanning system to precisely quantify the number of cells per slide. The cell-counting function, which was designed to determine the number of all nucleated (DAPI-stained) cells on the slide, allows an accurate counting of the tumor cells and the total number of cells analyzed in the given microscopic sample. The reliability of the cell-counting approach was demonstrated by the analysis of DAPI-stained images with 18-1,363 nucleated cells. A good correlation (r(2) = 0.965) between the manually and automatically gained results was observed. The counting accuracy could even be optimized after implementing a correction factor. To prove or disprove an interslide variation, routine bone marrow cytospin preparations from neuroblastoma patients were immunostained for GD2/FITC and counterstained with DAPI. Automatic cell counting of cytospin preparations from the same patients showed significant differences in the total cell number (up to 67% cell loss during preparation, with a maximum interslide difference of 4.7 x 10(5) mononuclear cells). We conclude that determination of the tumor cell content in hematopoietic samples is only reliable when it is performed together with accurate cell counting.
Assuntos
Neoplasias da Medula Óssea/patologia , Neoplasias da Medula Óssea/secundário , Contagem de Células , Microscopia de Fluorescência/métodos , Automação , Células da Medula Óssea/química , Imunofluorescência , Gangliosídeos/análise , Gangliosídeos/imunologia , Humanos , Processamento de Imagem Assistida por Computador , Neuroblastoma/patologia , Reprodutibilidade dos TestesRESUMO
We present the case of a 61- year-old black woman with a diagnosis of type 2 diabetes and a falsely elevated hemoglobin A1c (HbA1c) due to hereditary persistence of fetal hemoglobin. Physicians and allied health care professionals are alerted to this potentially significant problem in the diagnosis and management of diabetes mellitus (DM), particularly in the wake of the Diabetes Complications and Control Trial when "strict" glycemic control assessed by HbA1c is now the standard of care.
Assuntos
Diabetes Mellitus Tipo 2/diagnóstico , Hemoglobina Fetal/análise , Hemoglobinas Glicadas/análise , Diabetes Mellitus Tipo 2/sangue , Erros de Diagnóstico , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
Comparative genomic hybridization (CGH) has become a widely used method in molecular cytogenetics to screen for copy number aberrations in human malignancies. Although the hybridization protocol is relatively simple, the validation and quality control of CGH have remained difficult. We describe here a new modification of CGH, four-color CGH, which is based on conventional CGH with an added Cy5-labeled second reference DNA, that serves as an internal standard in every hybridization. The internal standard aids in identifying inconsistently hybridized chromosomal regions (such as 1pter, 19, 22). When using a special second reference DNA (from a sex-mismatched trisomy 13 cell line) for four-color CGH, it is possible to standardize the dynamic range of hybridization. The four-color CGH modification is simple to adopt, requiring only the addition of Cy5-labeled reference DNA to the existing hybridization protocol. The principles and the modifications of the CGH image analysis software are described in detail.
Assuntos
Hibridização de Ácido Nucleico/métodos , Neoplasias da Mama/química , Carcinoma/química , DNA de Neoplasias/análise , Feminino , Humanos , Hibridização in Situ Fluorescente/métodos , Controle de QualidadeRESUMO
A new multicolor-banding technique has been developed which allows the differentiation of chromosome region specific areas at the band level. This technique is based on the use of differently labeled overlapping microdissection libraries. The changing fluorescence intensity ratios along the chromosomes are used to assign different pseudo-colors to specific chromosome regions. The multicolor banding of human chromosome 5 is presented as an example.
Assuntos
Bandeamento Cromossômico/métodos , Coloração Cromossômica/métodos , Cromossomos Humanos/genética , Deleção Cromossômica , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 5/genética , Sondas de DNA/genética , Corantes Fluorescentes , Biblioteca Genômica , Humanos , Neoplasias/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Rare tumor cells circulating in the hematopoietic system can escape identification. On the other hand, the nature of these cells, positive for an immunologiCal tumor marker, cannot be determined without any genetic information. PROCEDURE: To overcome these problems a novel computer assisted scanning system for automatic cell search, analysis, and sequential repositioning was developed. This system allows an exact quantitative analysis of rare tumor cells in the bone marrow and peripheral blood by sequential immunological and molecular cytogenetic characterization. RESULTS AND CONCLUSIONS: In that virtually all tumor cells in a mixing experiment could be recovered unambiguously, we can conclude that the sensitivity of this approach is set by the number of cells available for analysis. Sequential FISH analyses of immunologically positive cells improve both the specificity and the sensitivity of the microscopic minimal residual disease detection.
Assuntos
Biomarcadores Tumorais/análise , Diagnóstico por Computador , Técnica Indireta de Fluorescência para Anticorpo , Gangliosídeos/análise , Perfilação da Expressão Gênica , Genes myc , Hibridização in Situ Fluorescente , Células Neoplásicas Circulantes , Neuroblastoma/patologia , Biomarcadores Tumorais/genética , Contagem de Células Sanguíneas , Exame de Medula Óssea , Aberrações Cromossômicas , Deleção Cromossômica , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 1/ultraestrutura , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 17/ultraestrutura , Sondas de DNA , Fluoresceína-5-Isotiocianato/análise , Corantes Fluorescentes/análise , Gangliosídeos/genética , Amplificação de Genes , Humanos , Hibridização in Situ Fluorescente/instrumentação , Indóis/análise , Neoplasia Residual , Neuroblastoma/genética , Sensibilidade e Especificidade , Trissomia , Células Tumorais CultivadasRESUMO
OBJECTIVE: To investigate the feasibility of various molecular forms of hemoglobin as markers for fetal nucleated red blood cells (NRBCs). METHODS: The presence of epsilon and gamma globin positive NRBCs was investigated in pure fetal blood and in blood from pregnant women before and after chorion biopsy. Maternal samples were enriched for NRBCs by various conventional methods, including limited enrichment by only positive CD71 selection or single density gradient. We searched for fetal cells on slides by automated scanning. Fetal cells were defined by (1) the presence of epsilon or gamma globin and (2) simultaneously by the presence of a Y chromosome signal. RESULTS: 18 of 25 gamma globin positive cells identified in blood samples after chorion biopsy were chromosome Y signal positive, and 1 cell had two X chromosome signals. 263 of 339 epsilon globin positive cells identified in blood samples after chorion biopsy were hybridized with X and Y chromosome probes. None had two X signals, and 249 were Y positive. In blood samples before chorion biopsy, only 1 epsilon globin positive fetal NRBC and no epsilon globin positive maternal NRBCs were found. CONCLUSIONS: Epsilon globin may be specific for fetal NRBCs. Only 1 epsilon globin positive fetal cell was identified in 1 of 12 blood samples before chorion biopsy, representing a total of 182 ml of maternal blood. This suggests that most fetal cells found in maternal blood by fluorescence in situ hybridization methods may not be NRBCs.