Development of a Potency Assay for Umbilical Cord Blood.

Total nucleated cell counts, cell viability, CD34 cell numbers and the colony-forming cell (CFC) assay constitute the main parameters used to characterize the quantity and quality of progenitors present in a unit of umbilical cord blood (UCB) prior to transplantation. Of these parameters, only the CFC assay provides information about the functional potential of progenitors. However, technical difficulties associated with the CFC assay have made it difficult to standardize this method among various UCB banks. Consequently, we are focusing on the development of a novel assay that relies on determining the growth potential of progenitors in response to cytokines by measuring intracellular ATP levels (HALO SPC-QC, Hemogenix). This assay is based on the principle that cytokine stimulated proliferation responses by hematopoietic progenitors are proportional to the production of intracellular ATP. To perform the HALO assay, cells obtained from leukocyte enriched UCB units are added to cytokine impregnated methylcellulose media and deposited in replicates of 6 into 96-well plates. After 7 days of culture, cells are lyzed to release ATP, which drives a luciferin/luciferase reaction producing bioluminescence. ATP levels for each well are measured in a plate luminometer and the assay is standardized using an ATP control. Since proliferation, not differentiation is measured, the assay is completed in 7 days rather than the 10–14 days required for CFC assays. For mononuclear cells or erythrocyte-depleted cord blood, ATP levels for cell concentrations as low as 750 cells/well can be detected. An intra-laboratory study comparing total mean ATP (μM) production/well (6 replicates) to the CFC counts from mononuclear cells from UCB, showed a correlation coefficient of 0.99 (n=6). An inter-laboratory study using aliquots from the same UCB to compare the HALO and CFC assays at 2 geographically separate sites revealed correlation coefficients of 0.90 and 0.34 (n=10), respectively. Together these data indicate that the HALO assay is capable of measuring the clonogenic potential of UCB progenitors. These data also demonstrate that highly reproducible results can be achieved by different testing sites when measuring ATP production from proliferating progenitors. Thus, we suggest that the HALO method may be more conducive to standardization among UCB banks. We also propose that the HALO assay may be better than the CFC assay for accurately and precisely measuring the potency of an umbilical cord blood unit prior to transplantation.