Comparison of Two Re-Freezing Methods on Previously Thawed Peripheral Blood Progenitor Cells.

Background: Peripheral Blood Progenitor Cells (PBPC) cryopreserved in DMSO are thawed at the patient’s bedside and infused immediately. Usually the infusions are completed without incident. There are occasions when mild/severe transfusion related reactions occur. When this does occur, the infusion is either slowed or paused for a short period of time. When reactions are severe enough to warrant stoppage of the infusion, transplant facilities maybe faced with discarding any remaining thawed cells. We have explored the question of salvaging the remaining cells by re-cryopreservation using two cryopreservation methods.

Study Design: Twenty PBPC were thawed in a 37C water bath until the internal temperature of the cells was approx 2-6C. The PBPC were not washed and allowed to remain at room temperature for 15 minutes. Ten PBPC were then placed on ice for 45 minutes. This simulated the transfer of cells to an off-site cryopreservation lab. These cells were then transferred to a new cryo bag, no change in the cryoprotectant solutions, and placed into a rate controlled freezer (RCF) bringing their temperature back to <−160C. The remaining 10 PBPC were allowed to remain at room temperature for an additional 10 minutes. The PBPC were then transferred to a −80C mechanical freezer. This simulated the transfer of cells to an internal laboratory without cryopreservation capabilities. All 20 PBPC were thawed after a minimum of 24 hours after return to their respective storage freezer conditions. We measured total nucleated cell count, viability by trypan blue, and CFU after the initial thaw and after the second thaw.

Results: The median viability of all PBPC after the first thaw was 69.5% (n=20; range= 35–83%). The median post thaw viability of the PBPC frozen by the RCF was 55.5% (n=10; range= 18–73%). Whereas, the median post thaw viability of the PBPC frozen in the −80C freezer was 25% (n=10; range 8–37%). Additionally, the median percent of viable cell loss from the initial pre-freeze data to the second thaw data for the RCF PBPC was 66.1% (n=10; range= 0 – 94.6%) and for the −80C PBPC was 94.1% (n=10; range= 91.3–97.8%. CFU growth was present for the RCF PBPC. No growth was demonstrated for the −80C PBPC.

Conclusion: Though neither method provided outstanding results, if faced with the dilemma of losing cells or attempting to save by re-cryopreservation, a transplant facility should consider RCF over −80C freezing.