Development of Multistage Magnetic Deposition Microscopy Pulak Nath, †,‡ Joseph Strelnik, †,§ Amit Vasanji, † Lee R. Moore, † P. Stephen Williams, † Maciej Zborowski, † Shuvo Roy, †,| and Aaron J. Fleischman* ,† Department of Biomedical Engineering/ND-20, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44195, and Department of Biomedical Engineering, University of Cincinnati, 2600 Clifton Avenue, Cincinnati, Ohio 45221 Magnetic deposition microscropy (MDM) combines mag- netic deposition and optical analysis of magnetically tagged cells into a single platform. Our multistage MDM uses enclosed microfabricated channels and a magnet assembly comprising four zones in series. The enclosed channels alleviate the problem plaguing previous versions of MDM: scouring of the cell deposition layer by the air-liquid interface as the channel is drained. The four- zone magnet assembly was designed to maximize capture efficiency, and experiments yielded total capture efficien- cies of >99% of fluorescent- and magnetically-labeled Jurkat cells at reasonable throughputs (10 3 cells/min). A digital image processing protocol was developed to measure the average pixel intensities of the deposited cells in different zones, indicative of the marker expression. Preliminary findings indicate that the multistage MDM may be suitable for depositing cells and particles in successive zones according to their magnetic properties (e.g., magnetic susceptibilities or magnetophoretic mo- bilities). The overall goal is to allow the screening of multiple disease conditions in a single platform. Isolation of targeted cells from a biologically relevant sample such as peripheral blood is a common requirement in biomedical science. Generally, cell separation is a sample preparation step where complex biological samples are simplified by depleting unwanted cells, thereby enriching desired cells. Separation is generally achieved based on the differences in their physical properties, such as density and size, or their biochemical proper- ties, such as surface antigen expression. 1,2 Advanced sorting techniques, such as fluorescence-activated cell sorting (FACS) 3 and magnetic cell separation, 4 have evolved over the years and now are capable of separating cells with high selectivity and recovery. Several disease conditions can be detected or monitored by counting the number of disease specific cells. For example, the low concentration of helper T lymphocytes (characterized by a surface expression of cluster of differentiation 4, or CD4+ cells) and a low ratio of the number of those cells to the number of cytotoxic T lymphocytes (CD8+ cells), CD4/CD8, is a clinical measure of AIDS progression. 5,6 The presence of circulating tumor cells (CTCs) is a hallmark of metastatic disease in cancer patients. The concentration of these cells in the blood has been shown recently to correlate with the prognosis following treatment of breast cancer. 7,8 A count of lower than 5 tumor cells in 7.5 mL of whole blood (that is, less than one CTC per milliliter of whole blood) has been shown to correlate with a lower incidence of relapse following chemotherapy. 7 Biological samples such as whole blood are a complex mixture of cells (∼5 × 10 9 /mL of red blood cells, ∼8 × 10 6 /mL of white blood cells, and ∼3 × 10 8 /mL of platelets) suspended in plasma. As a result, detecting the disease-specific cells in a blood sample for diagnostic purposes requires their isolation from the large pool of normal blood cells. Modern tools such as FACS and magnetic cell sorting rely on the interaction between cell surface antigens and antibodies conjugated to fluorochromes or magnetic particles, and therefore, these techniques can be very specific to targeted cells. 3,4,9 However, FACS machines are bulky and expensive and require special training to operate. Magnetic cell separation, on the other hand, is a simple technique that often employs a relatively small permanent magnet and a simple fluidic system to isolate magneti- cally tagged cells. Since virtually all untreated biological materials are diamagnetic or only weakly magnetic, magnetic cell separation can be highly specific and could be applied to cell mixtures as complex as whole blood. 10 Therefore, there have been significant advances in the area of magnetic cell separations. These develop- * Corresponding author. Phone: 1-216-445-3218. Fax: 1-216-444-9198. E-mail: fleisca@ccf.org. † Cleveland Clinic. ‡ Present address: Los Alamos National Laboratory, Los Alamos, NM 87545. § University of Cincinnati. | Present address: Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94158. (1) Recktenwald, D.; Radbruch, A. Cell Separation Methods and Applications; Marcel Dekker Inc.: New York, 1998. (2) Pertoft, H. J. Biochem. Biophys. Methods 2000, 44, 1–30. (3) Herzenberg, L. A.; Parks, D.; Sahaf, B.; Perez, O.; Roederer, M.; Herzenberg, L. A. A. Clin. Chem. 2002, 48, 1819–1827. (4) Safarik, I.; Safarikova, M. J. Chromatogr., B 1999, 722, 33–53. (5) Yu, L. M.; Easterbrook, P. J.; Marshall, T. Int. J. Epidemiol. 1997, 26, 1367– 1372. (6) Grodzinski, P.; Yang, J.; Liu, R. H.; Ward, M. D. Biomed. Microdevices 2003, 5, 303–310. (7) Cristofanilli, M.; Budd, G. T.; Ellis, M. J.; Stopeck, A.; Matera, J.; Miller, M. C.; Reuben, J. M.; Doyle, G. V.; Allard, W. J.; Terstappen, L. W.; Hayes, D. F. N. Engl. J. Med. 2004, 351, 781–791. (8) Cristofanilli, M.; Hayes, D. F.; Budd, G. T.; Ellis, M. J.; Stopeck, A.; Reuben, J. M.; Doyle, G. V.; Matera, J.; Allard, W. J.; Miller, M. C.; Fritsche, H. A.; Hortobagyi, G. N.; Terstappen, L. W. J. Clin. Oncol. 2005, 23, 1420–1430. (9) Shapiro, H. M. Practical Flow Cytometry, 4th ed.; John Wiley and Sons, Inc.: Hoboken, NJ, 2003. (10) Liberti, P. A.; Rao, C. G.; Terstappen, L. W. M. M. J. Magnet. Magnet. Mater. 2001, 301, 301–307. Anal. Chem. 2009, 81, 43–49 10.1021/ac8010186 CCC: $40.75  2009 American Chemical Society 43 Analytical Chemistry, Vol. 81, No. 1, January 1, 2009 Published on Web 12/04/2008