ABSTRACT
BACKGROUND: Extracorporeal photopheresis (ECP) is frequently utilized in the treatment of steroid-refractory acute and chronic graft-versus-host disease (GVHD). Although the mechanism of action is not fully understood, it has been postulated that its therapeutic effect is immunologic tolerance linked to the associated apoptosis of the treated cells. Despite significant advances in allogeneic hematopoietic stem cell transplantation (HSCT), prophylaxis and treatment of GVHD remain a challenge and major limitation associated with this therapy. Use of ECP is a valuable strategy; however, it is time, cost, resource intensive, and not readily accessible. OBJECTIVE: In an effort to expand access to this therapy, we are investigating the use of cryopreserved ECP-treated cells. This will provide the ability to administer a significant proportion of the treatment at a facility closer to the patient's residence, thereby decreasing the number of visits to the primary treatment center with the goal of improving and expanding access to this therapy. Here we report the effects of cryopreservation on ECP-treated leukocytes. STUDY DESIGN: Mononuclear cells were pheresed from human patients, ECP-treated, and collected for viability and apoptotic analysis. Cells were then cryopreserved at -80°C or -150°C for 1 week, 1 month, and 3 months. Following thaw, repeat viability and apoptosis studies were performed on the leukocytes. RESULTS: WBC viability for freshly ECP-treated leukocytes was 84.5% ± 3.5 at 1 week, 87.3% ± 5.2 at 1 month, and 79.1% ± 1.1 at 3 months post thaw. Similar results were seen for cells frozen in cryovials. Leukocytes frozen the day after ECP treatment had 1 week and 1 month WBC viabilities of 84.0 ± 4.1 and 83.1 ± 2.1, respectively. Apoptotic potential was well preserved at 3 months, with cryopreserved ECP-treated lymphocytes being 19.2%, 44.5%, 75.5%, and 94.0% apoptotic after thaw on days 0, 1, 2, and 3 in culture, respectively. CONCLUSIONS: ECP-treated leukocytes cryopreserved at -80°C or -150°C for 3 months remain viable and as capable of apoptosis as freshly treated cells. Cryopreservation of an ECP-product warrants further in vivo investigation as a strategy to facilitate access to this needed therapy.
Subject(s)
Graft vs Host Disease , Photopheresis , Humans , Cryopreservation , Graft vs Host Disease/prevention & control , Leukocytes , LymphocytesABSTRACT
OBJECTIVE: Major ABO incompatible hematopoietic progenitors from bone marrow (HPC(M)) donor collections that are destined for clinical transplantation are typically processed to deplete products of red blood cells (RBCs). The purpose of this study was to compare RBC depletion when using the Spectra Optia® relative to a 2-step method involving a COBE2991 instrument to obtain a buffy coat followed by a hydroxyethyl starch (HES) density gradient (COBE+HES) of the buffy coat. METHODS: Post-processing recoveries of products undergoing 4, 8, and 10 bone marrow processing (BMP) cycles (i.e. 1 cycle=1 volume of HPC(M)) with the Spectra Optia® were determined for volume, RBC content, viable total nucleated cells (vTNC), viable CD34+ cells (vCD34), viable CD3+ cells (vCD3) and colony-forming-cells (CFC). Subsequent RBC depletions with Spectra Optia® were then performed with 10 BMP cycles on additional HPC(M) collections and were compared against a retrospective analysis of historical COBE+HES post-processing data. RESULTS: Ten BMP cycles of HPC(M) (n=6) products were identified as optimal with volume reductions of 81.3±1.6 % and RBC reductions of 97.0±0.6 % with the Spectra Optia®. This also resulted in an average of 0.28 ±0.14 mL of RBC/kg (mean±SD; n=6) with vTNC yields of 65.0±10.9%, vCD34+ yields of 98.5±12.7%, and vCD3+ yields of 90.6±10.0%. Recoveries with the COBE+HES methodology resulted in vTNC recoveries of 62.9±20.5% (mean±SD; n=30) and 0.63±0.71 mL of RBC/kg (mean±SD; n=30). CONCLUSIONS: The Spectra Optia® is a viable option for depleting HPC(M) harvests of contaminating RBC in situations of ABO incompatibility. Target cells from a MNC rich fractionation were preserved through processing while eliminating RBC contaminants.
