Quantification of circulating cancer cells in whole blood samples by real time quantitative RT -PCR might be of clinical value for monitoring therapeutic effectiveness. In colon cancer patients, carcinoembrynic antigen (CEA) and cytokeratin 20 (CK20) have been frequently used for RT -PCR based tumor cell detection, but the specificity in particular for CEA has been questioned. In this study, we compared real-time RT -PCR for CEA and CK20 and analysed patients with metastatic disease (n=32) and healthy volunteers (n=17). CK20 mean values were elevated in cancer patients (P50.001) and defined a subgroup (38%) who showed CK20 levels at least 100-fold above the highest value of the healthy control group. In contrast, only two cancer patients (6%) showed elevated CEA levels. Samples of the healthy control group showed exclusively a CEA-PCR product of 798C melting temperature. Thirty per cent of the colon cancer patients showed an additional product of 828C melting temperature. The 828C product was identical with the amplification product of CEA-cDNA and cDNA from different colon cancer cell lines. Colon cancer cells were spiked into normal blood in 10-fold dilutions that resulted in a dose dependent shift of the melt curve from 798C to the 828C. Sequencing of the PCR products showed that white blood cells express a splice variant of CEA, which hinders detection of tumor cell cDNA in whole blood samples. Our findings have implications for the use of CEA as a diagnostic molecule (e.g. by RT -PCR). The discovery of a physiologically expressed CEA splice variant might lead to a better understanding of the biological function of CEA and its family members.

The use of a multi-step back flush procedure with a cord blood filter resulted in rapid enrichment of viable and functional monocytes from mononuclear apheresis units with significant reduction of contaminating platelets and red cells.