Quantitative Reverse Transcription-PCR Comparison of Tumor Cell Enrichment Methods

Abstract

The feasibility of detecting disseminated tumor cells through reverse transcription-PCR (RT-PCR) detection of tissue-specific genetic markers in extra-tumor compartments is currently unclear because of conflicting findings on assay sensitivity and specificity between studies [for reviews, see Refs. (1)(3)]. One potential source of these assay discrepancies includes the different sample preparations and analysis methods used between laboratories (4). Many studies have used nonenriched (NE) whole blood, although the high population of background cells within these preparations is considered to reduce RT-PCR sensitivity and specificity (5). To improve sensitivity and specificity, some investigators have used density-gradient enrichment (DGE) that is directed toward separating tumor cells in a mononuclear cell fraction from peripheral blood according to cell density (5)(6). Others have tested immunomagnetic separation (IMS) to isolate tumor cells with antibody-coated magnetic beads directed against epithelial cells (7)(8). The lack of standardized procedures and the paucity of quantitative analysis and of studies examining these methods in parallel have made the extent of the influence of sample-preparation methods difficult to determine. To assess the effect of sample preparation on the RT-PCR-based detection and quantification of disseminated tumor cells, we studied healthy donor blood samples with added colorectal cells. We used a newly developed, quantitative RT-PCR assay for the putative gastrointestinal epithelia-specific marker cytokeratin 20 (CK20) (9)(12) to evaluate the sensitivity, specificity, and quantitative PCR effects of DGE, two IMS techniques, and a NE preparation. For each series, 80 mL of peripheral blood were drawn from the antecubital vein of healthy volunteers with a Vacuette into heparinized tubes (Greiner). To avoid epithelial cells from skin puncture, the first 5 mL were always discarded. We added, per mL, 1000, 100, 10, 1, and 0 HT29 colorectal carcinoma cells to triplicate 5-mL blood aliquots. The 1000 and 100 cells/mL …