Washing Stem Cells Cryopreserved in Dimethyl Sulfoxide (DMSO) before Infusion Prevents DMSO-Related Side Effects with Little Loss of CD34 Positive Cells and Prompt Engraftment.

Abstract

Autologous peripheral blood stem cells are harvested and cryopreserved for weeks to months or even years in advance of the autologous peripheral blood stem cell transplant (PBSCT). The stem cells are cryopreserved in dimethyl sulfoxide (DMSO), a cryoprotectant agent that prevents ice crystals from forming and puncturing the stem cell membranes during the freezing process. Infused with the thawed cells, the DMSO can cause nausea and vomiting, headaches, seizures, renal insufficiency and stem cell death. We report on the results of 103 consecutive PBSCT, in which the DMSO was washed out before reinfusion. Viability, CD 34+ cell recovery as well as clinical endpoints such as time to engraftment (neutrophil and platelet counts) and infusion reactions for the 103 cases were assessed. The cryoprotectant solution used consisted of either 10% DMSO or a combination of 5% DMSO with 6% Hydroxyethyl starch. The stem cells were frozen using a rate-controlled freezer (Cryomed™ 1010) to −180°C and then stored in the liquid phase of liquid nitrogen (kept at approximately −190°C) for up to 4.7 years (mean duration of 42 days) following collection. The sample was thawed rapidly in a 37°C water bath and then washed manually with a washing solution of 0.9% Normal Saline (NS) and 10% anticoagulant citrate dextrose solution. After the sample was resuspended, it was centrifuged (RC-3, Sorvall™) and then suspended again in NS before infusion. Endpoints examined included CD 34+ cell recovery, viability, infusion reactions, and febrile episodes within 24 hours post-infusion, time to engraftment of platelets and neutrophils, highest serum creatinine (Cr) within 4 days following the transplant, and length of hospital stay. Automated WBC differentials were performed on a Beckman Coulter™ AcT Diff Analyzer and manually (when the WBC count was < 1000), using Turk's solution and a hemocytometer. Viability was assessed by exclusion of 2% Trypan blue solution. Mean CD 34+ cell recovery and viability were 85.4% and 79.38%, respectively. The mean serum Cr was 1.1. Time to neutrophil recovery was at day 12.4 (neutrophil count of 500 /uL) and platelet engraftment (defined as a platelet count of 20,000 /uL for three consecutive days without transfusion) was 14.9. Only 21% of patients experienced reactions during infusion, which included chills/rigors (9), fever (6), nausea/emesis (6), diarrhea (1), unusual body tightness or pain (3). There was also a case of mild hypotension during the infusion. 35 patients experienced neutropenic fevers during the 24-hour period post-infusion. All patients engrafted. Mean length of hospital stay was 19.4 days. In this study, fewer than 22% of the cases of PBSCT experienced infusion-related reactions. Most of these reactions were comparatively minor (chills/rigors). The percent CD 34+ cell recovery and viability were only slightly lower than historical infusions - done immediately after thawing, without washing out the DMSO. The data suggest that DMSO can be washed out of the stem cell product without notable loss of CD 34+ cells or a drop in their viability. Many DMSO-related side effects are, thus, eliminated.