Comparison of potency assays using different read-out systems and their suitability for quality control
ABSTRACT In this study, three of the most commonly used non-radioactive read-out systems for bioassays, the tetrazolium salt MTS/PMS, the fluorescent dye Alamar Blue and the ATP bioluminescence assay were compared regarding their suitability for quality control purposes. In this regard, three different potency assays using murine CTLL-2, as well as human DiFi and Kit 225 cells were performed. No major differences regarding accuracy and precision were detected between the different read-out systems. Both workload and hands-on time were similar for all three assays used. All read-out systems were suitable for use in quality control. However, luminescence and fluorescence techniques were much more sensitive than the colorimetric system. The first two could determine approximately ten times lower drug concentrations, and the assay could be performed by using considerably lower cell numbers. Moreover, in two of the three potency assays, the luminescence and fluorescence read-out systems provided higher signal to noise ratios leading to a higher precision of the assays. Regarding the comparison of the luminescence and fluorescence system, the ATP assay has the advantage to be much faster than the Alamar Blue assay. Consequently, in this study, the luminescence technique turned out to be the most advantageous of the three read-out systems.
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ABSTRACT: Rare hematopoietic stem cell populations are responsible for the transplantation engraftment process. Umbilical cord blood (UCB) is usually processed to the total nucleated cell (TNC), but not to the mononuclear cell (MNC) fraction. TNC counts are used to determine UCB unit storage, release for transplantation and correlation with time to engraftment. However, the TNC fraction contains varying concentrations of red blood cells, granulocytes, platelets and other cells that dilute and mask the stem cells from being detected. This does not allow the quality and potency of the stem cells to be reliably measured. 63 UCB segments and 10 UCB units plus segments were analyzed for the response of both primitive lympho-hematopoietic and primitive hematopoietic stem cells in both the TNC and MNC fractions. The samples were analyzed using a highly sensitive, standardized and validated adenosine triphosphate (ATP) bioluminescence stem cell proliferation assay verified against the colony-forming unit (CFU) assay. Dye exclusion and metabolic viability were also determined. Regardless of whether the cells were derived from a segment or unit, the TNC fraction always produced a significantly lower and more variable stem cell response than that derived from the MNC fraction. Routine dye exclusion cell viability did not correspond with metabolic viability and stem cell response. Paired UCB segments produced highly variable results, and the UCB segment did not produce similar results to the unit. The TNC fraction underestimates the ability and capacity of the stem cells in both the UCB segment and unit and therefore provides an erroneous interpretation of the of the results. Dye exclusion viability can result in false positive values, when in fact the stem cells may be dead or incapable of proliferation. The difference in response between the segment and unit calls into question the ability to use the segment as a representative sample of the UCB unit. It is apparent that present UCB processing and testing methods are inadequate to properly determine the quality and potency of the unit for release and use in a patient.Journal of Translational Medicine 12/2015; 13(1):434. DOI:10.1186/s12967-015-0434-z · 3.99 Impact Factor