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Salvage autologous hematopoietic stem cell transplantation (HSCT) is an effective treatment for patients with relapsed multiple myeloma (MM). Peripheral blood stem cells (PBSCs), a source of hematopoietic stem cells (HSCs), are collected before the first transplantation, and adequate quantities of PBSCs can be collected and stored potentially for years to support at least 2 transplantations for eligible patients. To ensure the safety of salvage HSCT in the treatment of patients in subsequent relapse, PBSCs must retain the potential to engraft even after several years of cryopreservation. Although PBSC viability has been studied extensively using in vitro techniques, few publications describe the most rigorous functional potency measure, of patients receiving a myeloablative conditioning regimen. This study describes a large single-institution experience evaluating the engraftment kinetics of PBSCs used in salvage transplantation after multiple years of storage compared with first transplantation for the same patients in the treatment of MM. A retrospective chart review of patients with MM undergoing HSCT between 2000 and 2021 identified 89 patients who received salvage autologous PBSCs stored for >1 year after first HSCT. PBSCs were cryopreserved and stored in vapor-phase liquid nitrogen refrigerators at ≤-150°C. All patients received a PBSC product from the same collection cycle for both transplantations. Differences in CD34 + cell doses and days to engraftment between the first and salvage transplantations were tested using the paired 2-tailed t-test and Wilcoxon signed-rank test. Univariate and multivariable linear regressions were used to determine the association between storage time and days to engraftment, adjusting for CD34 + cell dose and conditioning regimen in the multivariable model. The median duration of storage between the day of initial collection and salvage transplant was 5.4 years (range, 1.0 to 19.7 years). Engraftment kinetics demonstrated a sustained neutrophil engraftment (absolute neutrophil count >0.5 × 10 9 cells/L) at a median of 11 days after both the first and salvage transplantations (range, 8 to 15 days and 8 to 19 days, respectively; P < .05). The median time to sustained platelet engraftment (>20 × 10 9 cells/L without transfusion support) was 13.5 days after the first HSCT and 14 days after salvage HSCT (range, 9 to 27 days and 10 to 56 days, respectively; P = .616). After adjusting for CD34 + cell doses and conditioning regimens, there was no association between the duration of cryopreservation and days to neutrophil engraftment (r = 0.178, P = .130) or platelet engraftment (r = 0.244, P = .100). Engraftment kinetics of the salvage HSCT are comparable to those of the first HSCT even when products are stored in vapor-phase nitrogen refrigerators for a median of 5.4 years. There is no association between the duration of storage and time to engraftment when controlling for CD34 + cell dose and conditioning regimen. Prolonged storage of cryopreserved HSC products is a safe practice for MM patients undergoing salvage autologous HSCT., (Copyright © 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)