An Alternative Technique To Wash out DMSO from Thawed PBSC for Autotransplant.
Authors: Perotti Cesare , Fante Claudia Del , Viarengo Gianluca , Parisi Cristina , Marchesi Andrea , Bergamaschi Paola , Salvaneschi Laura 
Published: October 2019

Abstract
Introduction Dimethilsulfoxyde (DMSO) is a cryoprotectant routinely used for stem cell cryopreservation. At reinfusion, DMSO is toxic for the recipient and the risk of adverse effects (chills, flushing, nausea, vomiting, abdominal pain, chest tightness, blood pressure changes) are proportional to the DMSO amount. DMSO may also induce lifethreatening complications (cardiac and pulmonary arrest, acute renal failure) in low body weight patients or in subjects with a pre-existing cardiac disease. DMSO related complications are emphasized in “bad mobilizer” patients (CD34+/mL <20) in which multiple leukapheresis collections (large volume to reinfuse with high DMSO content) are needed to reach the stem cell dose necessary for transplant. The techniques (manual or automated) adopted to remove DMSO imply a various degree of stem cell loss, the risk of cell clotting and bacterial contamination.
Herein we present our experience in washing out DMSO from thawed PBSC with an alternative technique.
Material and Methods 48 PBSC bags, with a mean volume of 110 ml (range: 107–112) containing 10% DMSO were thawed for autotransplant in 8 patients affected with NHL-B (3), HL (3) and AML (2). 2 patients suffered from cardiac arrhythmia and 6 patients weighted less than 40 kgs (range 31–39.5). Each bag was thawed in a water bath at 37°C and immediately after was made a sterile connection with a bag containing a solution of the same volume (saline and 20% ACD-A) previously refrigerated at 4°C to minimize the DMSO cell damage. Afterward, the diluted PBSC bag was centrifuged at 1200g× 5mins at 4°C and the excess supernatant was removed using a plasma extractor. Finally, the cells were resuspended in an equal volume of refrigerated saline solution and human serum albumin (2%) and promptly reinfused to the patient. Pre-freezing, post thawing and post washing samples were collected to determine TNC, MNC, CD34+ cell content, viability and microbial contamination.
Results Pre-freezing, post thawing and post washing TNC, MNC, CD34+ cell content, viability and pre-freezing versus post washing cell recovery are shown in table 1. Data are expressed as mean and range (min-max). No microbial contamination was detected in all samples analyzed. No infusion related toxicity was recorded. No clotting was observed. No delay in WBC and PLT engrafment was documented.
Conclusions Our DMSO wash out technique is feasible, simple and safe and permits a satisfying recovery of MNC and CD34+ cells, preserving cell viability. PBSC manipulation at 4°C employing refrigerated solutions during every step mantains the quality of the thawed graft, minimizing DMSO stem cell damage with no delay in engraftment time. Finally, our method may be considered expecially when DMSO infusion could be lifetreathening in patients at high risk for a pre-existing cardiac, pulmonary or renal disease or in low body weight patients.
Table 1. Pre-freezing Post-thawing Post-washing Recovery (%) TNC (109) 46.2 (41.2–53.1) 40.4 (37.6–46.3) 31.7 (26.2–35.8) 67.3 (64–71.2) MNC (109) 24.5 (20.3–28.6) 22.4 (17.1–26.8) 20.3 (14.7–26.4) 83.3 (81–85.2) CD34+ (106) 35.7 (29–38.6) 31.3 (25–35.4) 27.2 (21.1–30.5) 77.1 (73.1–80) Viability (%) 98.9 (97.5–99.9) 87 (86.2–89.4) 79 (76.7–81.9)