Quality Control of Cell Processing and Cord Blood Storage Using HALO To Measure Proliferative Potential of Stem and Progenitor Cells.

Prior to the introduction of flow cytometry to detect and measure CD34⁺ cells in the early 1980s, quality control of stem cell transplants was performed using the granulocyte-macrophage colony-forming assay (GM-CFC). The major disadvantages of this growth potential assay are that progenitor and not stem cells are detected and the assay takes too long to perform. Yet, measurement of the growth potential of proliferating cell populations in vitro is the only technique that guarantees the presence of stem cells after cell processing and may predict engraftment and reconstitution. There are no assays presently that exhibit these characteristics in real-time. However, it is now possible to measure the proliferative potential of stem and progenitor cells after cell processing and/or storage in half the time required by the classical colony-forming assay (CFA). Originally developed for hemotoxicity testing for all stages of drug development, HALO (Hematopoietic/Hemotoxicity Assays via Luminescence Output) can be used as an in-line stem cell processing quality control procedure as well as a method to ensure the growth potential of processed and stored umbilical cord blood. HALO sensitively measures small numbers of proliferating cells and because cell differentiation is not detected, manual enumeration of colonies is not required. The assay is based on the CFA procedure. However, as cells proliferate in response to growth factors, there is a proportional increase in the intracellular ATP concentration. The assay is performed in a 96-well plate and release of intracellular ATP drives a luciferin/luciferase reaction to produce bioluminescence in the form of light measured in a plate luminometer. The kinetics of most hematopoietic proliferating populations, including mature stem cells, allows results to be obtained after 6–7 days in culture. Measurement of luminescence for all 96 well cultures takes about 5 minutes. An ATP standard dose response performed prior to sample measurement not only provides quality control of the reagents used, but intra- and interlaboratory comparison of results. Due to the rapid and easy set-up procedure with high-throughput capability, the proliferative capacity of several stem and progenitor cell populations from either single or large numbers of samples from bone marrow, peripheral blood or cord blood can be analyzed simultaneously. Rapid assay turnaround and multiparameter capability should therefore allow an increased safety factor for patients undergoing stem cell transplantation. Additional studies are underway to further validate this approach.