Experience in a Public Cord Blood Bank Using a Segment-Based Aldehyde Dehydrogenase Assay As a Biomarker for Umbilical Cord Blood Potency

Background: Cryopreserved cord blood units (CBU) provide a source of donor hematopoietic stem cells for transplantation for patients in need of a suitable donor.  Primary graft failures and delayed engraftment occur in up to 10-15% of patients following cord blood (CB) transplant which may be due to low potency, defined as low levels of viable engrafting hematopoietic stem and progenitor cells.  The majority of transplant centers currently select CBUs for their patients based on HLA match, pre-cryopreservation total nucleated cell count, and, in some centers, viable CD34⁺ content.  These methods do not account for potential insults caused by cryopreservation and thawing which may impact the overall potency.  We and others have shown that post-thaw hematopoietic colony forming units (CFU) measured on the transplanted product reflect potency and predict engraftment and survival.  However, post-thaw CFUs are not available at the time of CBU selection or transplantation because results are not available for 14 days.  CFUs measured on fresh CB correlate with the number of cells expressing high levels of the enzyme aldehyde dehydrogenase (ALDH^br), suggesting that ALDH^br content could be a biomarker for potency.  To test this, we retrospectively compared ALDH^brcontent of segments associated with transplanted CBUs and ALDH^brper kilogram correlated with engraftment.  Therefore, we developed a rapid ALDH-based potency assay in CBU attached segments that could be performed when HLA confirmatory typing (CT) was requested.

Methods: CBUs with attached segments banked at the Carolinas Cord Blood Bank and requested for CT between March 2010 and August 2013 were analyzed (n=2766).  Each segment was thawed and an aliquot of CB was taken for HLA CT.  The remaining CB was washed with 5% dextran/albumin to remove the DMSO.  Samples of 20,000 nucleated cells were plated in quadruplicate to quantitate CFUs. The number of viable (7-AAD^-), CD45⁺, ALDH^br, and CD34⁺ nucleated cells (glycophorin A^-) were measured by flow cytometry using multiparameter gating. 

Results: The number of ALDH^br (Spearman Correlation, r=0.81) and [ALDH^br viable CD34⁺] cells (r=0.73) both correlated well with CFUs measured on the segment (n=2730 for both analyses).  In contrast, CFU correlated less well with viable CD34⁺(n=2727, r=0.30).  These correlations were valid regardless of time in cryostorage.  Of the more than 2700 units assayed, more than 800 have been administered to patients. Thus, transplant outcomes data will be available to compare with laboratory metrics.

Conclusion: We developed a segment-based potency assay for CBUs selected for CT in a public CB bank.  Expression of ALDH alone and in combination with viable CD34⁺ best identified CBUs with high CFUs content and has potential for use as a biomarker of potency.  Viable CD34⁺ alone was not predictive of potency.  Further analyses correlating these results with engraftment are in progress.