Riv Ital Med Lab (2012) 8:26–35 DOI 10.1007/s13631-011-00…-… LETTER Isolation of circulating lung tumour cells using a non- EpCAM-based capture method Isolamento di cellule circolanti di tumore polmonare mediante separazione immunomagnetica Cecilia Bozzetti · Andrea Cavazzoni · Cecilia Carubbi · Prisco Mirandola · Pier Giorgio Petronini · Andrea Ardizzoni Ricevuto: 28 marzo 2012 / Accettato: 8 maggio 2012 © Springer 2012 C. Bozzetti () (E-mail: cbozzetti@ao.pr.it, Tel.: +39-0521-702676, Fax: +39-0521-995448), Andrea Ardizzoni Medical Oncology Unit, University Hospital, Via Gramsci 14, 43126 Parma, Italy A. Cavazzoni, P.G. Petronini Department of Experimental Medicine, Unit of Experimental Oncology, University of Parma, Parma 43126, Italy C. Carubbi, P. Mirandola Department of Anatomy, Pharmacology and Forensic Medicine, University of Parma, Parma 43126, Italy invading mesenchymal tumour cells lose cell–cell adhe- sion molecules [4–7]. With the aim of overcoming this issue, we separated cultured tumour cells in spiked peripheral blood samples from leucocytes and erythroid cells by immunomagnetic depletion using a non-EpCAM-based capture method. The non-small-cell lung cancer cell line Calu-3 that is known to show amplification of the HER2 gene [8] was used and CD45 and the glycophorin-A-negative immunophenotype was confirmed by flow cytometry. Cells were added to 5 ml of peripheral blood from healthy donors in amounts of 100 ± 15 cells and multiple recov- ery experiments were performed. After lysis of the red blood cells, the cell suspension was magnetically labelled with CD45 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD235A (glycophorin A) microbeads (Miltenyi) to deplete leucocytes and erythroid cells and loaded in an AutoMACS separator (Miltenyi). Columns were loaded and washed using the manufactur- er’s suggested protocol. The negative fraction was cen- trifuged and the pellet smeared on a glass slide. The cells were stained with May-Grünwald-Giemsa for cytomor- phological characterization and subjected to fluorescence in situ hybridization for HER2 assessment using the Path Vysion HER-2 DNA probe kit (Vysis-Abbott, Chicago, IL). The median recovery rate of Calu-3 cells obtained from 15 experiments was 20.2 % (standard deviation 11.5 %, range 8–44 %). The morphological features and HER2 amplification pattern of Calu-3 cells before and after immunomagnetic depletion are shown in Fig. 1. Keywords Circulating tumour cells · Lung cancer · Immunomagnetic separation It has recently emerged that detection and characteriza- tion of circulating tumour cells (CTCs) may provide important prognostic and predictive information in the treatment of lung cancer patients [1, 2]. In most studies, technologies used to capture CTCs have been based on the recognition of epithelial tumour cells by immunomag- netic anti-EpCAM antibody [1–3]. However, EpCAM is not expressed by all tumour cells. In fact, during the epithelial–mesenchymal transition process, which has been suggested by some as crucial for dissemination, 123 Bozzetti_Medicina di Laboratorio N.2 2012 08/06/12 14:57 Pagina 1