Evaluation of an Automated Ficoll Procedure To Enrich for Mononuclear Cells for Use in Clinical Trials.

Mononuclear cell fraction (MNC) enrichment from hematopoietic progenitor cells (HPC) cells is required for a number of clinical applications. Previously, we have used a manual method for enrichment of these cells. We evaluated an automated procedure for better consistency and patient safety (reduce risk of contamination). We have been using the Sepax (BioSafe) for the past 18 months to process Cord Blood (RBC depletion) before cryopreservation. Since the instrument can also do density gradient enrichment of mononuclear cells, we evaluated the ability to process different sources of hematopoietic progenitor cells (HPC) on the Sepax. Methods: Bone marrow, non-mobilized apheresis and cord blood were evaluated as a source of HPC. Briefly, 100ml of Ficoll solution were added to the chamber of the Sepax disposable. After initiating centrifugation, 50–110 ml of HPC were then overlayed onto the density gradient. The cells were fractionated, mononuclear cell fraction isolated, and then washed three times before final resuspension in 50ml of Saline/5% albumin. In some experiments the HPC were divided into two fractions so that a direct comparison of the Manual vs Automated enrichment procedure could be compared. Finally, a recent publication from Seeger et al (European Heart Journal28:766, 2007) suggested different density gradient solutions have an effect on MNC enrichment. Therefore, four different brands of density gradient solution (GE Ficoll-Paque Premium, Sigma Histopaque 1077, Accurate Lymphoprep and Lonza Lymphocyte Separation media) were evaluated to determine if there was a differential separation of subsets of cells. Results: The total nucleated cell (TNC), MNC and CD34 recoveries after MNC enrichment were similar for either bone marrow or cord blood HPC comparing the manual procedure and the automated procedure. The TNC and MNC recovery was higher for apheresis products (66% and 69% respectively) than the other two sources of HPC. This most likely reflects the enriched MNC starting population from an apheresis device. We evaluated the TNC and MNC recoveries over a range of initial TNC (7.5–20 × 108 cells) but could not see an effect of either cell number or cell concentration on recoveries. Nor could we see an effect of initial cell volume (50–100 mls) on recovery of cells post processing. Finally, we could not see a difference in either TNC or MNC recovery when the four different sources of density gradient solution were compared. Conclusion: We have shown that the Sepax instrument can be used for enrichment of mononuclear cells from HPC, Marrow, HPC, Apheresis and HPC, Cord. The benefits of an automated system are consistency, functionally closed system which reduces possibility of contamination documentation of run parameters and shorter processing time.

Her2/neu Expression by Reverse Transcriptase-Polymerase Chain Reaction in the Peripheral Blood of Prostate Cancer Patients

Background/Aims Evaluation of Her2/neu expression in the peripheral blood mononuclear cell fraction of prostate cancer patients by RT-PCR may afford an opportunity for the detection of circulating tumor cells and thus serve as a marker of micrometastatic disease. Methods We studied Her2/neu expression by reverse transcriptase-polymerase chain reaction in peripheral blood mononuclear cell fraction samples of 21 controls and serially in 43 patients with prostate cancer. Results None of the 21 controls expressed Her2/neu whereas 23.25% (95% CI, 11.75–38.63) of the patients were positive at entry into the study, and 65.11% (95% CI, 49.07–78.99) of them had at least one positive result during the follow-up period. Her2/neu positivity at study entry did not correlate significantly with PSA level, Gleason score, clinical stage or time to PSA progression. When we analyzed only patients with advanced disease, we observed a trend towards a shorter time to PSA progression in patients with at least one positive Her2/neu result during the follow-up (log-rank test, P = 0.08). Conclusions We conclude that Her2/neu expression in the peripheral blood mononuclear cell fraction of prostate cancer patients is frequent and therefore this assay may potentially be useful to detect the presence of micrometastatic disease in men with prostate cancer and for monitoring patients enrolled in trastuzumab-based therapeutic protocols.