CD34+ stem cell recovery after positive selection of "overloaded" immunomagnetic columns

Techniques for CD34+ cell enrichment of hematopoietic progenitor cells in grafts destined for transplantation of certain patients with the aim of lowering the amount of infused T lymphocytes and subsequently decreasing the risk of graft versus host disease (GVHD) have been well developed. Adaptations of these techniques should be useful for isolation of other phenotypically defined stem cells. However, a major limitation of techniques now available consists of the number of total nucleated cells or phenotypically defined stem cells that can be processed in a single procedure. Here, we show that recommended levels are much lower than the levels of cells that can be effectively processed by immunomagnetic sorting. Twenty-nine procedures were performed using the Clini- MACS (Miltenyi Biotec) device, which is recommended for processing <6x10(10 )total nucleated cells or <6x10(8) CD34+ cells. Procedures were divided in groups according to their total cellular or CD34+ cell content. We achieved a median CD34+ cell recovery of 68.60% with a median purity of 98.56%, regardless of the loading dose when samples possessing 2-10x10(10) total nucleated cells and 0.8-12.5x10(8) CD34+ cells were applied to a single column. The median levels of CD3+ cells and CD19+ cells in the final product were depleted by 5 logs and 3.8 logs, respectively; no differences were noted when the initial loading dose was increased. Moreover, we found no correlation between the total number nucleated or CD34+ cells loaded and the resultant CD34+ cell recovery. In conclusion, levels of both total nucleated cells and CD34+ can be processed in a single procedure with satisfactory and similar CD34+ cell recovery when these columns are loaded with up to two times as many cells as recommended.