Multicenter validation study on post-thaw viability testing of cryopreserved hematopoietic progenitor cell products

Post-thaw viability of cryopreserved hematopoietic progenitor cell (HPC) products is a critical quality attribute, yet testing practices vary widely across institutions. We conducted an international survey followed by a two-phase multicenter validation study to characterize current practices and assess inter-laboratory comparability using harmonized protocols.

In Phase 1, cord blood-derived HPC units were distributed to six centers for post-thaw viability testing. In Phase 2, cryopreserved HPC-apheresis cell product samples were distributed to three centers for viability assessment by flow cytometry under paired red blood cell lysis and non-lysis conditions at 0-120 min post-thaw. Center performance was evaluated using standardized deviation index (SDI) analysis.

Survey results revealed that 55.9% of respondents performed routine post-thaw viability testing, with considerable variability in methods (57.9% flow cytometry, 42.1% Trypan Blue) and acceptance thresholds (ranging from 40% to ≥90%). In Phase 1, center-level mean SDIs for CD34⁺ and CD45⁺ viabilities were generally within ±2 after outlier removal, indicating acceptable inter-laboratory consistency. In Phase 2, viabilities remained stable from 0 to 120 min post-thaw under both conditions.

This multicenter validation demonstrates that harmonized thawing and 7-AAD-based flow cytometry protocols can achieve good inter-laboratory comparability for cryopreserved HPC products. The data support SDI-based performance monitoring, pragmatic use of Trypan Blue where flow cytometry is unavailable, and operational flexibility for post-thaw testing within a 2-h window. These findings provide a foundation for consensus guidelines on post-thaw viability assessment.