BACKGROUND Collection and processing characteristics influencing quality of cord blood (CB) units play an essential role to cord blood banks (CBBs). At many CBBs, volume reduction is performed using hydroxyethyl starch (HES) and the Sepax (Biosafe) automated cell processing system. Due to the withdrawal of HES from the European market, a validation of the nonHES protocol was performed. METHODS This partially retrospective study identified CB characteristics such as gestational age and CB volume/cell count correlated with higher quality. For the nonHES validation, CB was analyzed for total nucleated cell (TNC), mononuclear cell (MNC) recovery, hematocrit (HCT) and colony-forming units (CFUs). Viabilities of CD34(+) and CD45(+) cells were determined by 7-aminoactinomycin D (7-AAD) and AnnexinV (AnnV) staining and compared for 21 mL and 42 mL buffy coat (BC) samples applying the HES/nonHES protocol. RESULTS Factors affecting the potency of CB transplants were the gestational age and the volume reduction to a defined BC volume. High initial cell counts and CB volumes correlated negatively with post-processing TNC recovery for lower BC volumes. Post-processing HES and nonHES results were comparable, but nonHES revealed a significantly lower post-thaw recovery of viable CD34(+) cells measured by 7-AAD/AnnV (21 mL: 45.4 ± 16.4%; 42 mL: 67.3 ± 14.5%) as compared with HES (21 mL: 72.7 ± 14.4%, P = 0.0164; 42 mL: 83.4 ± 14.7%, P = 0.0203). CONCLUSIONS Due to the lower post-thaw CD34(+) cell viability (AnnV(-)/7-AAD(-)) for nonHES samples, the use of HES is recommended, ideally combined with a high BC volume. The post-processing HCT has no statistically significant impact on the post-thaw CD34(+) cell viability (AnnV(-)/7-AAD(-)).