Measurement of Stem Cell Potency, Assessment of Acceptance Limits for Release Criteria and Prediction of Umbilical Cord Blood Engraftment Using a Replacement for the Colony-Forming Cell Assay
Authors: Rich Ivan N. , Hall Karen , Harper Holli , Freed Brian M 
Published: October 2019

Abstract
The total nucleated cell count (TNC) is a major factor in the acceptance or rejection of an umbilical cord blood (UCB) unit. Yet it is stem cell potency that will decide whether a UCB unit has the capability to engraft and repopulate the patient after transplantation. The colony-forming cell (CFC) assay has been employed in a retrospective manner in order to document the possibility of growth potential of stem cell products destined for transplantation. Although some centers count and differentiate colonies, many use the assay to document either growth or no-growth because the results are difficult to interpret and provide little predictive value. Subjectivity and lack of an external standard to which the assay can be calibrated and validated means that the CFC assay can neither be employed as a reliable and reproducible stem cell potency assay, nor can it be used to define release criteria of UCB products for transplantation. A cell potency and release assay is required by standards and regulatory organizations. Viability and CD34 may be important for engraftment, but cannot measure stem cell potency. In a recent article in Transfusion (48:620, 2008), Reems et al describe a novel instrument-based, ATP bioluminescence proliferation assay used for UCB and compared at two geographical locations. Results demonstrated a correlation for the assay between the locations with a correlation coefficient of R=0.94 (p<0.001; r2=0.89). In addition there was a correlation between the ATP and CFC assays (R=0.79). Additional results have demonstrated a direct correlation between intracellular ATP (iATP) concentration and total colony counts with a correlation coefficient (R) of greater than 0.97, thereby validating the ATP assay against the CFC assay and allowing the former to replace the latter. The ATP assay has now been further developed into a stem cell potency and release assay using a UCB reference standard (RS). In this study, the engraftment results of 56 frozen UCB sample pellets tested was known. Of the 36 samples that could be tested at 3 cell doses (sufficient cell numbers) for CFC-GEMM against the UCB RS, 13 exhibited a potency ratio greater than 1, indicating a greater stem cell potency, while 9 exhibited a potency less than 1. All 13 samples also exhibited an iATP concentration greater than 0.1μM, and with 1 exception, all samples engrafted. A second group of 14 samples demonstrated a range of iATP concentrations between 0.05 and 0.1 μM iATP and also engrafted. A third group of 28 samples had iATP values between 0 and 0.05μM. Within this group, 14 did not engraft. The other 14 samples engrafted, but on the basis of the iATP concentration, would not have been predicted to engraft. In summary, there are 4 conclusions. First, stem cell potency values greater than 1 can be correlated with engraftment, while those lower than 1 would have questionable engraftment potential. Second, the steeper the slope of the UCB cell dose response, the greater the proliferation potential and the greater the probability of engraftment. Third, samples exhibiting iATP values between 0.05 and 0.1μM or greater, represent present acceptance limits for release of the UCB sample for transplantation and engraftment would be predicted. Lastly, from the present study, the ATP assay has an engraftment prediction rate of 71%. These results clearly demonstrate that the new assay not only can replace the CFC assay, but has the capability of providing significantly more meaningful information than has been possible.