Qualitätskontrolle in der Transplantation hämatopoetischer Progenitorzellen : Optimierung und Standardisierung der Testung klonogenen Wachstums
A rapid and reliable method based on viable CD34+ cells for evaluation of colony growth after cryopreservation of hematopoietic progenitor cell grafts J. Fasold, A.Ketels, R. Repp, M. Gramatzki, A. Humpe Division of Stem cell and Immunotherapy, Second Department of Medicine, Schleswig-Holstein University Hospital Campus Kiel, FRG Purpose: Quality control before and after cryopreservation of hematopoietic progenitor cell grafts (HPC) varies between different centers and still lacks standardization. Especially evaluation of clonogenic growth after cryopreservation is associated with considerable variations of the applied protocol and of the reference parameter, i.e. CD34+ cells, mononuclear cells, or CD45+ cells. We evaluated a method involving a minimum of manipulations of the sample before plating the cells and normalizing for viable CD34+ cells (series 1, S1) or for viable CD45+ cells (series 2, S2). Methods: Satellite tubes of 563 autologous HPC grafts with a concentration of viable CD34+ cells/µL ranging from 30 to 28,220/µL satellite tubes were ranked for the CD34+ cell concentration and divided into 30 sections. From each of the sections one sample (range of CD34+ cell concentration: 140 – 12,710/µL) of different patients was chosen on a random basis for analysis. Cells were thawed rapidly in a 37 °C water bath and diluted depending on cellular concentrations at least 1:10 in IMDM. Cell suspension were further diluted with a final DMSO concentration of less than 0.1% in the assay and plated in quadruplicate with 200 viable CD34+ cells per dish and in parallel with 5x10E+04 viable CD45+ cells per dish. Dishes were incubated at +37°C under fully humidified atmosphere and 5% CO2. Colonies were counted after 14 days of culture under a dissection microscope. Aggregates containing more than 20 cells were considered colonies. Results: In S1 all assays were countable. A median number of 47 CFU-GM (17 – 82)/200 viable CD34+ cells were counted. In S2 only 14 samples were analyzable with a median number of 48 CFU-GM (9 – 153)/dish. The other 16 samples were due to an excess of colonies not countable. In S1, the median standard deviation for the number of CFU-GM in each of the 30 analyzed samples was 8% (1.5 – 21%). One sample was analyzed 24-times with a coefficient of variation of 18.8%. Conclusions: The presented method only involving dilution steps but no washing steps or further manipulation procedures before analysis of CFU-GM growth is comparable to the situation at the time of infusion at the bed-side of the patient. The normalization for 200 viable CD34+ cells leads to consistent colony growth results independent of the concentration of CD34+ cells in the original graft.
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