VIABILITY OF FRESH, STORED AND CRYOPRESERVED HAEMOPOIETIC PROGENITOR
CELLS FOR TRANSPLANTATION
*Loretta Huckstepp1,
1 Department of Haematology (Stem Cell Transplant Laboratory), 2
Department of Immunology (Flow Cytometry), Sydpath, St Vincent's Hospital
Sydney, Australia
Adequacy of Haemopoietic Progenitor Cell (HPC) harvest for transplantation is based on the number of CD34+ cells collected. Routine storage and cryopreservation techniques can damage these cells, reducing the viable transplant dose. The number of colony forming units (CFU) is an effective but time-consuming method to assess graft viability. Another method is the trypan blue dye exclusion assay, which reveals non-viable cells in the total nucleated cell (NC) population. A reliable and rapid indicator of CD34+ cell viability has been sought, using flow cytometry to assess the viable CD34+ cell population. Peripheral blood HPC apheresis samples were tested immediately, after 72 hours 4°C storage (n= 17), and/ or after cryopreservation in 10% DMSO (n= 22). Analysis by flow cytometry including 7AAD assessed viable CD34+ and CD45+ populations. This new method was compared with trypan blue dye exclusion and 14-day CFU assays. Linear regression analysis compared these rapid methods with the clonogenic assays CFU-GM, CFU-GEMM, CFU-E and BFU-E. Viable CD34+ cells/ml correlated significantly with CFU-GM/ml, CFU-GEMM/ml, and total colony number (p=<0.0001). Total CD34+ cells/ml showed a significant but weaker correlation with all colony types. No correlation was found between the CFU and the NC viability assays. CD34+ cells were consistently more viable than nucleated cell viability in the fresh, stored and cryopreserved samples, and the viable CD34+ correlation with CFU-GM and CFU-GEMM was consistent in each sample environment. The nucleated cell concentration had no significant effect on the viability of CD34+ or total nucleated cells stored at either 4°C or post cryopreservation (p = 0.168, p= 0.094 respectively). Viable CD34+ cell enumeration is a rapid and cost-effective method of assessing graft quality. Preliminary data indicate viable CD34+ cell enumeration may be used as an alternative to the established CFU assay in clinical settings.