Improved cord blood thawing procedure enhances the reproducibility and correlation between flow cytometry CD34+ cell viability and clonogenicity assays

Umbilical cord blood (UCB) is cryopreserved and stored in cord blood banks (CBBs) for allogeneic transplantation in pediatric and adult patients [ [1] ]. It is known that cord blood unit (CBU) cell viability is deeply affected by both cryopreservation and thawing processes [ 2 , 3 ]. Cell viability is determined by flow cytometry using 7-Aminoactinomycin D (7-AAD) before and after cryopreservation. However, some authors report that the obtained data may be inaccurate when performing post-thawing assays due to membrane instability and cellular unspecific uptake of 7-AAD [ [4] ]. It is possible that this phenomenon varies during the staining protocols and, consequently, affects the flow cytometry results. On the other hand, clonogenic efficiency (CLONE; percentage of effective colonies from a known number of seeded CD34+ cells) is a complementary assessment to flow cytometry and provides information about cell viability, lineage commitment and proliferation ability. Therefore, it may be a more suitable quality control of the unit. The correlation between CD34+ cell viability, as assessed using flow cytometry and CLONE, is a subject under research and may depend on several pre-freezing variables [ 5 , 6 ]. We hypothesize that if the thawing and staining procedure-associated biases are removed, a real correlation factor may be determined.