( for immunomagnetic enrichment. ...)Sensitive detection of tumor cells in,,,peripheral blood of carcinoma patients,,,by a reverse transcription PCR method

for immunomagnetic enrichment.

Lower detection limit
Cell spiking experiments with different cell lines were performed to test the potential sensitivity of the assay. After inoculation and immunomagnetic enrichment, two carcinoma cells were detected in 5 mL blood samples (1010 cells) using the AdnaTest CancerDetect kit (figure 3). In addition specificities of up to 100 % are obtained for each marker of the multiplex PCR. We demonstrated this using blood of healthy donors, patients with different non-malign diseases (e.g. gastrointestinal diseases) and inoculated non-malignant epithelial cells. No signals were detected in the PCR analysis (data not shown). The combination of the two steps above provides high specificity and sensitivity for the diagnostic approach.

RNA stabilization
Due to the high instability of cellular RNA in vitro, preserving the RNA expression profile is essential for reliable analysis of gene expression. To achieve this we developed a reagent, which stabilizes cellular RNA for up to 2 days at 5 C and 45 C, and up to 8 days at room temperature (figure 4). Storage at 45 C caused a reduction in expression level especially of tumor marker 2, but the tumor marker could still be detected. Thus we are able to overcome the critical aspect of degradation of mRNA during storage and transport of patient samples.

Clinical validation
At present, the clinical validity of the diagnostic system is being investigated in several studies for each tumor entity (colorectal, testicular and breast cancer). Exemplary we investigated 50 colorectal carcinoma patients at the time of primary diagnosis and in followups over a period of six months.
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