Optimization of a Method for CD34-Selection of Cryopreserved Peripheral Blood Hematopoietic Cells

Introduction CD34-selections can minimize Graft Versus Host Disease (GVHD) in Hematopoietic stem cell transplantations (HCT) and boosts (HCB). Selections are most often done on fresh products; however, CD34-selections on cryopreserved hematopoietic progenitor cells (HPC) carries many logistical benefits. We optimized a method for CD34-selection on thawed, cryopreserved HPC apheresis collections (HPC-A). Methods CD34-selection on cryopreserved versus fresh products requires modification; this primarily regards cell-free DNA resulting from the freeze/thaw cycle. Thawed products were pooled together and diluted 1:1 in 3 equal steps with Thawing Buffer (5% LMD/2.5%HSA supplemented with DNAse and magnesium chloride). The pellets were resuspended with Washing Buffer (CliniMACS PBS/EDTA buffer containing 1% HSA and supplemented with citrate, DNAse, and magnesium chloride) and directly treated with concentrated DNAse and magnesium chloride. Processing followed standard procedure except for using modified Washing Buffer. If clumping or gelling was noted, the products were again treated with concentrated DNAse and magnesium chloride prior to filtering to remove clumps. Once placed on the CliniMACS for selection, the standard Selection Buffer (CliniMACS PBS/EDTA Buffer with 1% HSA) was used. Results We achieved viable CD34-positive cell recoveries of ∼70% in trials. Post-thaw CD34-selection of HPC of nine patients achieved a median CD34 recovery of 57.5% (range: 41.3-76.6%) and a median CD3 Log reduction of 4.76 (range: 4.32-5.14), comparable to prior selections on nineteen fresh HPC(A) using mixed-effects analysis (Table 1). No significant difference was apparent in either the CD34 recoveries or the CD3 log reductions. Conclusion kCoordinating a fresh selection can prove challenging. Stored cells may offer an economical and logistically favorable approach. CD34-selection on post-thawed apheresis products proves to be a viable option. Our method of post-thaw selection is feasible and yields comparable cell recoveries to selections on fresh products. This technique may offer greater access of selected products to patients for whom fresh CD34-selections are not possible.