Relationships Among Commonly Used Measures of Cord Blood Potency, ALDH^br Cell Content, and Colony Forming Cell Content in Cord Blood Units Prior to Cryopreservation: Towards An Improved Metric for Potency of Banked Cord Blood,

Despite adequate total nucleated cell (TNC) dosing, graft failure and engraftment delays remain a significant issue for patients undergoing cord blood transplantation (CBT). Previously, we have shown that recovery of colony forming units (CFU) after cord blood unit (CBU) thaw is superior to TNC and CD34⁺ content in predicting engraftment. However, the CFU assay requires weeks and is not standardized. Aldehyde dehydrogenase (ALDH), an enzyme expressed in stem and progenitor cells (ALDH^br cells), is detected by a functional, flow-based intracellular assay and can be completed within a few hours. In this study, we determined the statistical relationships among TNC, CD34⁺, ALDH^br cells and CFU in a large cohort of fresh CBUs prior to cryopreservation with the ultimate goal of defining the best parameter(s) to predict potency defined by successful engraftment. Methods: As part of routine banking procedures, post-processing TNC, mononuclear cell count (MNC), CD34⁺cells and CFU content were enumerated on fresh CBUs donated to the Carolina Cord Blood Bank (n=5268) or identified for directed donation (n=14) between 9/07–7/09. ALDH^br cell content was measured using Aldecount (Aldagen, Inc.). Correlations among these various CBU characteristics were determined. Results: Of the 5282 fresh CBUs analyzed, the median values for TNC, CD34⁺, MNC, ALDH^br cells and CFU content were: 11.8×10⁸ (range, 2.9–55.5×10⁸), 3.36 x10⁶ (range, 0.17–98.2×10⁶), 5.5×10⁸(range, 1.7–20.0×10⁸), 4.3x 10⁶ (0–36.6×10⁶) and 34.0×10⁵ (range, 0.6–193.3×10⁵), respectively. There were significant correlations (p<0.0001) among all of the cord blood (CB) parameters measured as presented in Table 1. In standardized, univariate modeling which allowed for ranked comparisons of the CB parameters using AIC values, CFU was best predicted by TNC (AIC=11,501) followed closely by ALDH^br (AIC=12,012) and, to a lesser degree, MNC (AIC=12,412) and CD34⁺ (AIC=12,612). In multivariate modeling of CB parameters (Table 2), results were similar to the standardized univariate analysis.

In this large dataset of fresh CBUs, the parameters measured (TNC, MNC, CFU, CD34⁺ and ALDH^br) are well correlated and predictive of each other. Furthermore, total ALDH^br measured on fresh CB was well correlated with other CB parameters, including CFUs that also assesses viable cells and is the unit characteristic that best predicts engraftment. Given the technical difficulties associated with measuring CFUs, ALDH^br content has promise as a potential CFU surrogate as a potency assay for CBUs. ALDH^br measurements were modestly, but significantly more reliable in predicting CFU content than CD34⁺ or MNC content in this cohort of fresh CBU. We and others have previously shown that different types of cells survive freezing and thawing of the CBU with different efficiencies. Consequently, we are studying which CBU parameter best correlates with engraftment after thawing of CBUs or from segments attached to CBUs in order to improve assessment of potency during the process of CBU selection for CBT.

Balber:Aldagen, Inc: Consultancy, Equity Ownership.