Within-unit paired aliquots reduce between-unit variation and help distinguish freeze–thaw injury from storage-duration effects in cryopreserved cord blood products

Background Cryopreserved cord blood is an established source for hematopoietic stem cell transplantation, and banks store units for years to decades before release. How long a unit retains conforming viability and hematopoietic capacity is therefore tied to release eligibility decisions. However, most stability studies compared different units across storage durations, confounding storage effects with between unit variability. Methods In this quality control program, the same cord blood unit was aliquoted, cryopreserved, and thawed by storage duration; pre-freeze and thawed specimens were compared in a paired manner so each unit served as its own control. From 67 units manufactured from 2012 to 2024, we obtained 229 pre-freeze–thaw pairs (storage up to 13 years 2 months). We assessed total nucleated cell (TNC), CD34 + count, colony-forming capacity (CFU), viability with trypan blue (vTNC-TB), and three 7-AAD based flowcytometric viabilities (vTNC, vCD45+, vCD34+), using paired Wilcoxon tests and linear mixed-effects models with measurement-era adjustment. Results After a single freeze–thaw cycle, core engraftment related indicators declined: vCD45 + by ~ 18% points, vCD34 + by ~ 12, and CD34 + count by ~ 20%. CFU was ~ 14% lower overall but not significantly reduced in the short-storage first-thaw subset (recovery 0.95, p = 0.339). TNC was largely preserved (recovery 0.98, p = 0.036), as was vTNC-TB (~ 1.6-point decrease). Most injury occurred at the initial freeze–thaw. After adjusting for the measurement-era confounder, no significant additional deterioration with storage was found over the observation period (up to 13 years). vTNC-TB read ~ 6% points higher than vTNC. Conclusions Within 13 years, the principal quality changes in cord blood were associated with the first single freeze–thaw cycle rather than with long-term storage, although storage effects could not be fully separated from measurement era. For long-term cryopreserved human cells such as cord blood products, a standardized quality-control program managing external changes such as measurement instruments is needed.