CD34+ cell content for selecting umbilical cord blood units for cryopreservation

Worldwide, more than 220,000 umbilical cord blood (UCB) units have been HLA-typed and stored in public banks, and more than 6000 UCB transplants have been performed in children and adults. Although criteria for selecting collections suitable for cryopreservation are not standardized, most UCB banks require a total nucleated cell (TNC) content of at least 60 ¥ 10 to 100 ¥ 10 for storage. We qualify UCB units for freezing if they have a TNC content that is at least 100 ¥ 10. The CD34+ cell content, however, has been shown to influence engraftment and survival of UCB transplants. If UCB grafts contain at least 1.7 ¥ 10 CD34+ cells per kg, there is a high probability of survival in recipients. Based on this observation, we recommend that UCB banks consider this criterion for storing UCB units for transplantation. Retrospectively, we analyzed the CD34+ cell content of 2150 UCB units containing initial TNC of at least 100 ¥ 10 and stored in Valencia Cord Blood Bank for 5 years. CD34+ cells were quantified by flow cytometry. Cord blood (5 ¥ 10 cells) was incubated using monoclonal antibodies (MoAbs) conjugated with CD34 fluorescein and CD34 phycoerythrin (Becton Dickinson, San Jose, CA) and 7-aminoactinomycin D as marker of DNA staining. Flow cytometric analysis was performed with computer software (CellQuest, Becton Dickinson, Franklin Lakes, NJ). We compared results using progenitor cell enumeration kits (Procount, Becton Dickinson) with our standard protocol in 25 cases and found similar results. The mean CD34+ cell percentage and total content, after volume reduction and before cryopreservation, were 0.36 0.22 percent and 38.09 ¥ 10 29.81 ¥ 10, respectively. Only 488 UCB units (22%) contained less than 20 ¥ 10 CD34+ cells and 159 (7%) had a CD34+ cell content less than 10 ¥ 10. Previously, we reported that the CD34+ cell content before freezing represented the hematopoietic stem cell content of thawed and washed UCB units. To optimize UCB selection, we performed CD34+ cell counts before and after volume reduction of 70 UCB units. The mean CD34+ cell percentage and total CD34+ cell content before and after volume reduction were 0.28 0.14 percent and 50.74 ¥ 10 41.64 ¥ 10 and 0.30 0.15 percent and 41.65 ¥ 10 34.7 ¥ 10, respectively. Mean CD34+ cell recovery after volume reduction was 82.17 14.94 percent. We found a significant correlation between white blood cell (WBC) content and percentage of CD34+ cells (Fig. 1 and Table 1). We are considering the inclusion of the initial CD34+ cell count as an additional criterion for selecting UCB units for cryopreservation. To increase the CD34+ cell content of stored UCB units, we propose a cutoff of 20 ¥ 10 for the number of CD34+ cells. In fact, the CD34+ cell content of transplanted UCB units is usually more than 20 ¥ 10. If we were to apply this criterion for 1 year in our program (2000 UCB collections, 600 UCB stored containing initial TNC of 100 ¥ 10), we would perform approximately 463 additional CD34+ tests (increased cost of 5666 USD). In contrast, we would not store 137 (22%) of the units (decrease cost of 87,680 USD). This approach would result in a substantial increase in the percent and content of CD34+ cells in our stored UCB units (0.43 0.21% and 48.28 ¥ 10 29.39 ¥ 10), resulting in more UCB units available for patients weighing 11 to 28 kg. In addition, we would save 82,014 USD, which would allow us to include more collaborating maternity wards. Pilar Solves, MD, PhD e-mail: solves_pil@gva.es