RESUMEN
AIMS: To develop and evaluate an automated method for quantification of HER2 fluorescence in situ hybridisation (FISH) signals. METHODS: Using a popular, open source image manipulation tool, ImageJ, a macro for FISH signal assessment was created. A comparison against traditional manual counting was performed in breast cancer specimens from 42 patients. The tumour specimens were hybridised with probes for HER2 and chromosome 17 centromere (CEP17) and selected areas were digitised for image processing. Hybridisation signals were calculated both manually and automatically with the ImageJ custom macro. RESULTS: The correlation coefficient between the automatic and manual HER2/CEP17 ratios was 0.98. The corresponding percentage agreement was 90% and the kappa value was 0.82. CONCLUSIONS: This study shows that it is possible to automate the determination of HER2 amplification by the use of open-source software, with results comparable to manual counting. The automated counting decreases the time needed for sample analysis and provides possibilities to enhance inter- and intralaboratory reproducibility of results. The FISH quantification tool (FishJ) is available for download as an ImageJ macro or alternatively it can be utilised through a web interface with an option of uploading FISH images for hybridisation signal counting. Combined with digitisation of FISH samples, the FishJ macro enables gene copy number to be assessed and re-evaluated on any area of a digitised specimen.
Asunto(s)
Algoritmos , Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Procesamiento Automatizado de Datos , Genes erbB-2 , Procesamiento de Imagen Asistido por Computador , Hibridación Fluorescente in Situ/métodos , Área Bajo la Curva , Recuento de Células , Centrómero/ultraestructura , Aberraciones Cromosómicas , Cromosomas Humanos Par 17 , Femenino , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Humanos , Sensibilidad y EspecificidadRESUMEN
The aim of the present study was to assess the T cell cytokines IFN-gamma, IL-2, IL-4 and IL-5 in labial salivary glands (LSG) in Sjögren's syndrome (SS) and healthy controls using RT-PCR and immunohistochemistry. IFN-gamma is always or almost always produced in SS and in healthy controls. IL-2 was also found in some samples, but IL-4 and IL-5 were not. Less than 2% of all inflammatory mononuclear cells contained immuoreactive IFN-gamma or IL-2. Cytokine mRNA profile in LSGs in SS is skewed towards a T(H)1 pattern. The classical T(H)1 cytokines are also produced in normal glands, even in the absence of foci. T(H)1 type response may play an active role as part of the mucosal associated lymphoid tissue/responses, perhaps in prevention of reactivation of latent viruses. This may also make the exocrine glands a locus minoris resistentiae when the self tolerance is broken.
Asunto(s)
Interferón gamma/genética , Interleucina-2/genética , Glándulas Salivales/inmunología , Síndrome de Sjögren/inmunología , Células TH1/inmunología , Elementos sin Sentido (Genética) , Secuencia de Bases , Expresión Génica/inmunología , Humanos , Interleucina-4/genética , Interleucina-5/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A foreign-body-type host response can contribute to the induction and release of collagenolytic tissue-destructive enzymes of pathogenetic significance. Our aim was to analyse collagenase-3 in two conditions with putative involvement of foreign-body reactions. Synovial membrane-like tissue samples were obtained from cases of aseptic loosening of a total hip replacement (THR) and osteoarthritis (OA). The reverse transcription polymerase chain reaction (RT-PCR) disclosed that all the samples from patients contained collagenase-3 mRNA compared with only three out of ten control samples. The identity of the RT-PCR amplification product was confirmed by nucleotide sequencing. Immunohistochemical staining showed that collagenase-3 was present in endothelial cells, macrophages and fibroblasts, including those found in the synovial lining. This finding was confirmed by avidin-biotin-peroxidase complex-alkaline phosphatase-anti-alkaline phosphatase double staining and the specificity of the staining by antigen preabsorption using recombinant human collagenase-3. Collagenase-3 was released into the extracellular space and thus found in the synovial fluid in all patient samples as shown by Western blotting. The similar extent of collagenase-3 expression in aseptic loosening and OA compared with the low expression in control synovial membrane suggests involvement of a similar, foreign-body-based pathogenetic component in both. Comparative analysis of collagenase-3 and of foreign particles indicates that paracrine factors rather than phagocytosis per se are responsible for the induction of collagenase-3. We suggest that due to its localisation and substrate specificity, collagenase-3 may play a significant pathogenetic role in accelerating tissue destruction in OA and in aseptic loosening of a THR.
