Proc. to reduce the number of normal blood cells in a malignancy patient’s blood from 4.05 109 to 8.04 103 cells/ml and still recover, on average, 2.32 CTC per ml of blood. For all of the malignancy patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per ml of blood, with a range of 282 to 0.53 CTC per ml of blood. Unlike a majority of other published studies, this study focused on quantifying as many factors as you possibly can to facilitate both the optimization of the process as well as provide information for future overall performance comparisons. The authors are not aware any other reported study which has achieved the overall performance reported here (a 5.76 log10) in a purely unfavorable enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final, enrich product contains CTC’s or other cell type relevant to the specific Dinaciclib (SCH 727965) question (i.e. does the CTC have predominately epithelia or mesenchymal characteristics?). recognized by their morphology and by ligand-specific monoclonal antibodies conjugated with fluorescent, or brightfield, dyes. Surface markers as well as intracellular markers can be used as a target antigens. The Dinaciclib (SCH 727965) advantage of immunocytochemical approach is usually tumor cells are actually observed under a microscope; however, a disadvantage is that it is challenging to find tumor cells among non-tumor cells at a frequency of 1/105 and lower (Braun and Pantel, 2001). In circulation cytometry, tumor cells are recognized by size, granularity and fluorescent signals emitted by fluorescently labeled antibodies targeting specific cell surface markers. Several commercially available reagents are available that target surface antigens common to many CTC (which are commonly from epithelial origin), and through specific fixation procedures intracellular markers such as cytokeratins can also be stained (Chosy et al. 2003; Green et al. 2000; Brandt et al. 2001; Paterlini-Brechot and Benali 2007). While circulation cytometry analysis can provide multi-dimensional information (multiple cellular attributes can be detected per cell), it is a time consuming technique once the quantity of cells to be analyzed becomes greater than 106 cells. In addition, unless the cells are sorted after identification, the cell is usually discarded. An alternative to fluorescent sorting is usually to pre-sort the cells using magnetic enrichment technology and then analyze the cells with a circulation cytometer (Racila, et al. 1998). Numerous studies have shown that tumor cells can be detected using RT-PCR technology targeting specific mRNA characteristic of malignancy cells such as: Cytokeratin 8, 19, carcinoembryonic antigen (CEA), epidermal growth factor receptor, EGFR, and for melanoma markers such as tyrosinase, MUC18, glycoprotein gp100/pmel17, and MART1/Melan-A (Paterlini-Brechot and Benali, 2007; Mocellin et al. 2006). Sensitivity of this RT-PCR method can be high, especially when the nested PCR technique is used. However, the results in general are more qualitative than quantitative, and poor specificity is commonly reported (Ko et al. 2000). A possible explanation for the variance in performance is that the expression of the so-called tumor-specific genes in a normal NESP cell is not zero but near zero. Therefore, a large number of normal cells expressing, at a low level, this tumor specific marker, could make a sample appear made up of a tumor cell (i.e. produce a false positive condition). This phenomenon has been called the illegitimate expression of a gene and has been previously explained for CK19 (Ko et al. 2000). In an attempt to begin to quantify the sensitivity of RT-PCR for CTC markers in human blood, Tong et al. (2007) quantified the number of cells and the amount of mRNA for EGFR needed for a positive detection using RE-PCR in normal, human peripheral blood spiked with Head and Neck Squacous Cell Carcinoma cells for three different cell lines. It was previously reported by Zen et al. (2003) that EGFR is usually a potential maker for CTC of human, oral squamous cell carcinoma. As might be expected, the amount of mRNA for EGFR expressed by the three different lines varied, significantly. Consequently, to positively identify mRNA for EGFR in a spiked blood sample, the final concentration of a malignancy cell in blood at the Dinaciclib (SCH 727965) time of cell lysis ranged from 1 malignancy cell in 1,000.