Evaluation of An Automatic Washing Method for DMSO Cryopreserved Peripheral Blood Hematopoietic Progenitors Using the Sepax S-100.

Dimethylsulfoxide (DMSO) containing cryoprotective solutions are routinely used for the storage of hematopoietic progenitors (HP). At room temperature, DMSO is toxic for the cells and may produce severe adverse reactions during their infusion, especially in the pediatric patients. These problems can be avoided by washing the cells prior to the infusion. Our objective was to test if an automatic washing method (Sepax S-100, Biosafe) allowed us to preserve the CD34+ cell numbers with an adequate viability and engraftment potential.

Forty five peripheral blood HP apheresis that have been cryopreserved using autologous plasma plus 9% DMSO were studied. After rapid thawing in a water bath at 37° C, an automatic wash with the Sepax S-100 (2 washes cycle) was performed. Nucleated cell levels determined by an hematology analyzer, flow cytometry CD34+ cell counts and Trypan Blue cell viability test were performed on aliquots collected prior to and after the washing technique. The paired Student's t-test and the Pearson's correlation coefficient were used for the statistical analysis.

The mean total nucleated cell (TNC) and CD34+ cell recovery was 75,47% ± 3, and 94,66% ± 4,62 respectively. In spite of the TNC significant loss (p<0,001), there were no significant differences between the pre and post-washing CD34+ cell numbers (p=0,08) or viability (p=0,19). The TNC loss had no correlation with the pre or post-washing number of platelets (p=0,18 and p=0,16 respectively), neither with the loss of platelets during the procedure (p=0,31). In contrast with the 40% of untoward reactions recorded in our historical data of 226 DMSO containing cell infusions, we observed just one adverse effect (2,22%) with the washed cells. One patient reported a transient mild abdominal pain with nausea and one vomit. Median time to neutrophil engraftment (>500 cells /mL) and platelet engraftment (>50.000 cells/mL) were 11 ± 0,2 and 20 ± 1,4 days respectively.

The Sepax S-100 automatic wash protocol of DMSO containing peripheral blood progenitor cells determines a good CD34+ cell recovery and preserves their viability and engraftment potential. This method avoids the DMSO infusion related adverse events and it constitutes a closed and easy to do procedure.

No relevant conflicts of interest to declare.