Molecular Detection of Circulating Prostate Cells in Cancer II: Comparison of Prostate Epithelial Cells Isolation Procedures

Abstract

The sensitive and specific detection of circulating tumor cells holds great promise for more accurate staging of cancer patients. Several reverse transcription-PCR (RT-PCR) procedures based on tissue-specific mRNA expression are now able to detect one cell derived from a given tissue among >10 peripheral nucleated blood cells [PNBCs; for a review, see (1)]. However, even for a single marker, highly discrepant results have been observed among the available clinical studies; e.g., the frequencies of positive prostate-specific antigen (PSA) RT-PCR results range from 25% (2) to 80% (3) in patients with metastatic prostate cancer (CaP), blurring the clinical relevance of these assays. Standardization and quality control in molecular diagnosis are crucial to the solution of this issue. We have previously studied factors potentially affecting RT-PCR results (4), and this current work focuses on the approaches for harvesting prostate cells among nucleated blood cells. Since the first 1992 clinical report describing the RT-PCR detection of circulating prostatic cells in CaP, the majority of reported assays have used gradient separations to recover nucleated cells from the peripheral blood (1). We (5) and others (6) rather choose overnight hypo-osmotic red blood cell lysis as an easier and more cost-effective protocol. Because the approach used to harvest PNBCs may account for discrepancies in clinical results, we have compared a panel of nucleated blood cell separation methods. Fresh blood from four healthy blood donors (total volume, 400 mL) was sampled in 40 × 10-mL EDTA-treated tubes (Becton-Dickinson). The LNCaP cell line, derived from a metastatic prostate carcinoma, was cultured as described (7). After trypsinization (2.5 mL/L trypsin), 10 confluent LNCaP cells were added into each of the 40 tubes, which were gently mixed. Six tubes were processed immediately by overnight hypoosmotic red blood cell lysis, as described (5)(6): Two volumes of ammonium chloride (9 …