Presence of circulating tumor cells (CTC), as detected by the CellSearch 1 System, in patients with metastatic carcinomas is associated with poor survival prospects. CellTracks TDI, a dedicated image cytometer, was developed to improve the enumeration of these rare CTC. The CellSearch System was used to enumerate CTC in 7.5 mL blood of 68 patients with cancer and 9 healthy controls. Cartridges containing the fluorescently labeled CTC from this system were reanalyzed using the image cytometer, which acquires images with a TDI camera using a 403/0.6 NA objective and lasers as light source. Automated classification of events was performed by the Random Forest method using Matlab. An automated classifier was developed to classify events into CTC, apoptotic CTC, CTC debris, leukocytes, and debris not related to CTC. A high agreement in classification was obtained between the automated classifier and five expert reviewers. Comparison of images from the same events in CellTracks TDI and CellTracks Analyzer II shows improved resolution in fluorescence images and improved classification by adding bright-field images. Improved detection efficiency for CD45-APC avoids the classification of leukocytes nonspecifically binding to cytokeratin as CTC. The correlation between number of CTC detected in CellTracks TDI and CellTracks Analyzer II is good with a slope of 1.88 and a correlation coefficient of 0.87. Automated classification of events by CellTracks TDI eliminates the operator error in classification of events as CTC and permits quantitative assessment of parameters. The clinical relevance of various CTC definitions can now be investigated. '

Characterization of rare cells usually requires high sensitivity quantification of multiple parameters. Detection of morphological features of these cells is highly desired when routinely identifying circulating tumor cells (CTC) in blood of patients. We have designed an image cytometer intended for fast and sensitive routine analysis of CTC. After an initial scan, prospective events can be revisited for more detailed analysis. The image cytometer features: 375, 491, and 639 nm laser lines, a 403/0.6NA objective, a CCD camera operating in TDI mode, servo stages to move the sample in two dimensions and a piëzo microscope objective positioner to move the objective in the third dimension. ImageJ is used for dedicated image analysis. A homogeneous illumination area, measuring 180 3 180 lm 2 , was created by the use of a rotating diffuser in combination with two micro-lens arrays. For feed-forward automatic focusing of the sample during a scan, a 3D spline was fitted through 30 predetermined focus positions before scanning the sample. Continuous signal acquisition is made possible by using a CCD operating in TDI mode synchronized to the movement of two servo scan stages. The limit of fluorescence sensitivity is 120 PE molecules on a bead with a diameter of 6.8 lm, at a scanning speed of 1.0 mm s 21 . The resolution of the imaging system is 0.76 lm in the TDI scan direction at a wavelength of 580 nm. Identification of cells is facilitated by scatter plots of the fluorescent parameters in which each individual event can be viewed for its morphological features by fluorescence as well as bright field. The image cytometer measures quantitative fluorescence and morphological features at a high sensitivity, high resolution, and with minimal overhead time. It has the ability to relocate events of interest for further detailed analysis. The system can be used for routine identification and characterization of rare cells. ' 2011 International Society for Advancement of CytometryKey terms cell analysis; image cytometry; TDI; rare event detection; CTC FLOW cytometry (FCM) is the standard technology for characterization and enumeration of cells in a heterogeneous cell mixture. It is routinely used for diagnosis and monitoring of diseases that result in specific changes of a particular cell population. Yet, for characterization of rare populations FCM has its limitations. This explains the emergence of alternative cell analysis technologies for dedicated applications (1-4).We aim to routinely analyze and enumerate tumor cells that circulate at extremely low concentrations in the blood of cancer patients. We started by using FCM as the analysis platform (5-7). The variable background of the events that classified as tumor cells in a multidimensional gate in FCM (5-7), observed in blood of normal donors urged us to use morphological confirmation. Fluorescence microscopy in combination with immunomagnetic enrichment of circulating tumor cells (CTC) proved to be the right combination. It removes the background and identifies ...

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