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ISAC XX Abstracts on Disc Presented by Purdue University Cytometry Laboratories |
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| Cell analysis system based on immuno-fluorescent analysis of immuno-magnetic selected and aligned cells with Compact Disk technologies. |
Arjan G.J. Tibbe1, Bart G. de Grooth1, Leon W.W.M Terstappen2, G.J. Dolan2, J. Greve1
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| Abstract Number: 6009 Established Technologies – Other Established Technologies |
| A schematic representation of the novel cell analysis method, Cell Tracks, is shown in Figure 1. Whole blood is incubated with ferromagnetic nanoparticles labeled with monoclonal antibodies as well as fluorescent-labeled monoclonal antibodies. After incubation the blood mixture is placed in a specially designed chamber that is placed between two wedge-shaped magnets. The optically transparent uppersurface of the chamber contains ferromagnetic lines of nickel (Ni) deposited by lithographic techniques. The magnets create a gradient moving magnetically labeled cells up-wards to the top of the chamber. When they reach the top of the chamber, they become subject to a high local internal gradient induced by the Ni lines and align in between them. The cells that are not magnetically labeled slowly move down under the influence of gravity. In this way, the cells labeled by the immuno-magnetic particles are well aligned at the uppersurface of the chamber (Fig 2). A 635nm laser-diode is focussed on the Ni lines through an objective of a common compact disk player. A feedback system using the laser light reflected from the Ni lines was developed to keep the laser focussed on a line of cells while the chamber is moved in the Y-direction. After one line is scanned, the laser jumps to the next line and the chamber is moved in the opposite Y-direction. By moving the chamber the cells pass under the laser focus one after each other. For each cell the fluorescence signals generated are analyzed and the cell position recorded. To demonstrate that the system can indeed measure relevant cell populations, blood was incubated with CD45-labeled magnetic particles; APC-labeled CD4 monoclonal antibodies and Oxazine750, which stains leukocytes differentially. Both dyes can be excited by the 635nm laser-diode and their emissions can be spectrally separated. Figure 3 shows the scatter plot of Oxazine750 versus CD4-APC; CD4+ lymphocytes, CD4- lymphocytes, monocytes, eosinophils and neutrophils appeared in clearly distinct positions. The absolute as well as relative frequency of these cell populations correlated well with those obtained from a hematology analyzer and flowcytometer. This cell analysis method is significantly less complex than current cell analysis equipment and provides additional functionality through its ability to subject cells to repeated and varied analysis while they remain in their natural environment, i.e., whole blood. |
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| Keywords: Cell Tracks; magnetic selection; Oxazine750; Compact Disk; HeNe; Array |
