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Rosso F.,Surgical and Emergency science | Papale F.,Surgical and Emergency science | Barbarisi A.,Surgical and Emergency science
Methods in Molecular Biology | Year: 2012

Immunogold labeling (IGL) technique has been utilized by many authors in combination with scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to obtain the identi fication/ localization of receptors and antigens, both in cells and tissues. Environmental scanning electron microscopy (ESEM) represents an important tool in biomedical research, since it does not require any severe processing of the sample, lowering the risk of generating artifacts and interfere with the IGL procedure. The absence of metal coating could yield further advantages for our purpose as the labeling detection is based on the atomic number difference between nanogold spheres and the biological material. Using the gaseous secondary electron detector, compositional contrast is easily revealed by the backscattered electron component of the signal. In spite of this fact, only few published papers present a combination of ESEM and IGL. Hereby we present our method, optimized to improve the intensity and the speci ficity of the labeling signal, in order to obtain a semiquantitative evaluation of the labeling signal. In particular, we used a combination of IGL and ESEM to detect the presence of a protein on the cell surface. To achieve this purpose, we chose as an experimental system 3T3 Swiss albino mouse fibroblasts and galectin-3 ©Springer Science+Business Media, LLC 2012.

Lieto E.,Surgical and Emergency science | Galizia G.,Surgical and Emergency science | Orditura M.,F. Magrassi A. Lanzara' | Romano C.,The Second University of Naples | And 6 more authors.
Oncology Letters | Year: 2015

The present study evaluated the presence and clinical relevance of a cluster of differentiation (CD)26+/CD326-subset of circulating tumor cells (CTCs) in pre-and post-operative blood samples of colorectal cancer patients, who had undergone curative or palliative intervention, in order to find a novel prognostic factor for patient management and follow-up. In total, 80 colorectal cancer patients, along with 25 healthy volunteers were included. The easily transferable methodology of flow cytometry, along with multiparametric antibody staining were used to selectively evaluate CD26+/CD326-CTCs in the peripheral blood samples of colorectal cancer patients. The multiparametric selection allowed any enrichment methods to be avoided thus rendering the whole procedure suitable for clinical routine. The presence of CD26+/CD326-cells was higher in advanced Dukes' stages and was significantly associated with poor survival and high recurrence rates. Relapsing and non-surviving patients showed the highest number of CD26+/CD326-CTCs. High pre-operative levels of CD26+/CD326-CTCs correctly predicted tumor relapse in 44.4% of the cases, while 69% of post-operative CD26+/CD326-CTC-positive patients experienced cancer recurrence, with a test accuracy of 88.8%. By contrast, post-operative CD26+/CD326-CTC-negative patients showed an increase in the three-year progression-free survival rate of 86%, along with a reduced risk of tumor relapse of >90%. In conclusion, CD26+/CD326-CTCs are an independent prognostic factor for tumor recurrence rate in multivariate analysis, suggesting that their evaluation could be an additional factor for colorectal cancer recurrence risk evaluation in patient management. © 2015, ONCOLOGY LETTERS. All right reserved.

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