Thawed and Washed Apheresis Products Keep an Excellent CD34+ Cells Viability for 24 Hours Using Either Voluven or Normal Saline Plus Albumin
Authors: Blanquer Blanquer Miguel , Alguero Carmen , Menchon Pilar , Ferrer Assumpta , Martínez Avilés Pilar , García Hernandez Ana , Iniesta Francisca , Moraleda Jose Maria 
Published: October 2019

Abstract
Cryopreservation of products rich in progenitor cells is mandatory for the feasibility of autologous hematopietic progenitor cells transplants. The most used product nowadays is the apheresis of mobilized peripheral blood mononuclear cells. However, cryopreservation implies the use of cryoprotectant molecules, such as DMSO, that can be toxic for the patients. Moreover, the thawing of the product goes with some unavoidable cell death and liberation of cytoplasmic content such as cytokines to the medium. And so, adverse reactions during the product infusion are not infrequent. Our group has demonstrated that washing the thawed apheresis products both with Voluven or with normal saline plus 5% albumin (NSA) is able to almost completely avoid infusion reactions without losing CD34+ cells. Here we wanted to know whether it also had the benefit of extending CD34+ viability for 24 hours after washing.
METHODS: We thawed 3 spare peripheral blood mononuclear cells apheresis products that had been cryopreserved with 9%DMSO. Ten mL of each product were separated and the remaining volume was splitted in 2 bags to be washed either with Voluven or NSA. Sepax 2 smartwash automatic program was used to wash the cells. The washed cells were stored at 4ºC and a sample was taken and immediately analyzed at 0h, 1h, 2h, 4h and 24h. At the same time points 10 mL of each bag were separated and kept 30 min. at room temperature (RT) before being analyzed. A blood count was performed on all the samples. Flow cytometry was used to measure CD45+ and CD34+ cells viability by 7AAD staining.
RESULTS: The mean CD45+ cells viability was 69% after thawing the cells ,81% immediately after washing them with Voluven, and 81%, 80%, 79% and 73% 1h, 2h, 4h and 24h after the wash. When using NSA the mean CD45+ viability was 79%, 92%, 93%, 92%, 93% and 87% at the same time points. When the samples were kept for 30 additional minutes at RT, the mean CD45+ viability was 78%, 81%, 84%, 82%, 84% and 81% with Voluven, and 80%, 92%, 94%, 91%, 95% and 86% with NSA. Regarding CD34+ cells, when washed with Voluven the mean viability was 46%, 87%, 87%, 92%, 83% and 69%, while it was 61%, 91%, 91%, 92%, 89% and 84% when NSA was used. The mean CD34+ cells viability results after 30' at RT for each time point were 79%, 90%, 82%, 91%, 82% and 81% with Voluven and 85%, 90%, 90%, 94%, 93% and 86% with NSA. Moreover the mean viable CD34+ cells recovery at 24h was 90.94% for Voluven and 87,88% for NSA.
CONCLUSIONS: We have obtained an excellent stability of the CD34+ cells viability for 24h after washing the mobilized mononuclear cells apheresis products both with Voluven and with NSA when the products are stored at 4ºC. Moreover, the viability is not affected when the cells are kept for an additional 30' at RT. The viable CD34+ cells loss at 24h was scarce.

Disclosures
Blanquer Blanquer: Pfizer: Research Funding.